Final segregation of male meiotic products in plants

ABSTRACT

The invention provides a plant in which, by virtue of modulation of the expression of a TETRASPORE (TES) or TES-like (TLK) gene, or of modulation of the activity of a protein encoded by such a gene, tetrad formation in the anther is disrupted, or is capable of being disrupted, such that callose cross-wall formation in the tetrad fails to the extent that fusion between two or more of the four microspore nuclei may occur.

FIELD OF THE INVENTION

[0001] The present invention is in the field of plant biotechnology.More specifically, it pertains to techniques by which crosses betweenplants of different taxa are made possible. This is made possible by thecloning and characterisation of the TETRASPORE (TES) gene, whosesequence is provided herein.

BACKGROUND OF THE INVENTION

[0002] Plant Genome Ploidy

[0003] Plants differ from animals in that they exhibit a range of genomeploidy. Animal somatic cells are usually diploid, and animal gameteshaploid. Whilst many plants also conform to this, others are, forexample, tetraploid (and thus their gametes are diploid) or hexaploid(and thus their gametes are triploid). For example, wheat is generallyhexaploid. In some cases, genome ploidy varies within a single species,e.g. in certain ornamental plants, and also within the model speciesArabidopsis.

[0004] This poses an important problem for plant breeders. It isdifficult to perform crosses between plants of different genome ploidiesand most such crosses fail. In some cases, it is possible to altergenome ploidy by tissue culture. Normally, small explants (pieces ofleaf or stem) are cultured in vitro in the presence of colchicine whichinhibits the mitotic spindle. The cells then undergo another round ofDNA synthesis without cell division. The colchicine is then removed fromthe culture medium allowing the cells to progress into cell division.With suitable care and attention, it is possible to generate tetraploidand hexaploid plants by this method. However, this is extremelytime-consuming, as it involves generating a parent plant with thedesired parental characteristics but altered somatic cell ploidy.

[0005] Genome Ploidy in Pollen Development and Fertilisation in Plants

[0006] In plants, pollen carries the male gametes (sperm) to the femaleones (ova). In the anther of the male parent plant, pollen developmentbegins with the formation of a tetrad of microspores by meiosis. Theseare the progenitors of the male gametic cells. In, for example, adiploid plant, the somatic cells are diploid and the microspores arehaploid. In the normal tetrad, the four microspores are separated fromone another by callose cross-walls. Each microspore then undergoes afurther series of mitotic divisions, leading to a male gamete cellcontaining two sperm nuclei and a vegetative nucleus. All of these arehaploid. Pollen containing such cells is released from the male parentplant and fertilises the female parent plant.

[0007] During this process, one of the sperm nuclei fuses with thehaploid ovum to form a diploid cell generally equivalent to an animalzygote. However, the other sperm nucleus fuses with a diploid “centralcell” of the female parent plant to form a triploid cell. This is not afeature of the reproductive process in animals. Divisions of thetriploid cell generate the endosperm, which provides resources for thegrowing embryo in the developing seed.

[0008] In the above, it has been assumed that the plant has diploid (2n)somatic cells, thus haploid (1n) gametes, diploid central cells and atriploid (3n) endosperm. In tetraploid (4n) systems, the gametes arediploid, the central cells tetraploid and the endosperm hexaploid. Inhexaploid systems, the gametes are triploid, the central cells hexaploidand the endosperm nonaploid (9n).

[0009] It can therefore be seen that the normal ratio of genomecontributions in the endosperm is 2 maternal: 1 paternal. In mostplants, Arabidopsis being something of an exception, this “endospermbalance number” is crucial to successful seed development. This is whycrosses between plants of different genome ploidies generally fail.

[0010] Clearly, it would be beneficial to the plant breeding industry tohave available more straightforward methods for generating genomes of arange of different ploidies in the gametes of parent plants.

SUMMARY OF THE INVENTION

[0011] We have cloned the TETRASPORE (TES) gene of Arabidopsis. Itssequence is provided herein.

[0012] We have previously determined that the product of the TES gene isrequired for male meiotic cytokinesis (Spielman et al, Development 124,2645-2657 (1997)). In that paper, we described four mutant alleles ofthe TES gene and noted that, following failure of male meioticcytokinesis in the mutants, all four microspore nuclei remain within thesame cytoplasm, with some completing their developmental programmes toform functional pollen nuclei. The meiotic division seen in normalpollen development takes place in TES gene mutants. In other words, theTES gene appears to be necessary for the formation of the callosecross-walls in the tetrad that normally separate the four microsporenuclei. We have also previously investigated parent-of-origin effects onseed development in Arabidopsis (Scott et al, Development 125, 3329-3341(1998)).

[0013] We have now cloned the TES gene and investigated its properties.As discussed above, tetrads in TES gene mutants lack the callosecross-walls that normally separate the four mircospore nuclei. Theresult of this is that very large pollen grains containing all theproducts of meiosis are formed. Strikingly, these meiotic products go onto develop relatively normally to form sperms and vegetative cells.However, we have now determined that fusion sometimes takes placebetween the microspore nuclei resulting in the formation of “male germunits” of differing ploidy. As discussed above, a male sperm nucleuswould normally be haploid in a diploid plant, diploid in a tetraploidplant and triploid in a hexaploid plant. However, in TES gene mutantplants, sperm nuclei with higher ploidy are sometimes formed.Importantly, the ploidy of male germ units in TES gene mutants is alsoheterogeneous such that, for example, some may be haploid, some diploid,some triploid and some tetraploid in Arabidopsis.

[0014] This has important implications in plant breeding, because itwill facilitate wide crosses within and between species wherein the maleand female parent plants have different genome ploidies. Essentially,because of the variation in the ploidy of the sperm generated in plantsin which the product of the TES gene is suppressed or inactivated, thereis a good chance that the ploidy of some of the sperm will conform tothe ploidy of the female parent plant, thus ensuring correct balancenumbers for the zygote and, crucially, the endosperm of the resultantembryo. This offers a much more convenient way of performingmixed-ploidy crosses than is currently available.

[0015] Significantly, TES gene mutant plants (tes) also seem to beunaffected in the female development, so there is no reason to believethat manipulating the TES gene will compromise female fertility. TESgene mutants show reduced fertility, presumably because of the overalllack of congruence with the ploidy of the female parent plant. However,this is substantially outweighed by the advantage of being able togenerate a range of sperm ploidies to facilitate crossing. In addition,when TES protein activity is suppressed, e.g. by the use of antisense orRNAi techniques, such suppression can be rendered inducible so that thevariability of sperm ploidy can be switched on and off as desired.

[0016] The TES gene has been identified by positional cloning. The TESgenomic sequence extends for some 10 Kb, and the cDNA encodes a proteinof 932 amino acids. Importantly, sequence comparisons positivelyidentify the product of the TES gene as an N-terminal motor kinesin,containing a tropomyosin domain. RT-PCR expression analysis reveals TESmRNA to be expressed in all parts of the Arabidopsis plant throughoutthe life cycle reflecting a level of redundancy also found in manykinesins. Strikingly, database searches have identified a second kinesinwith TES-like features (named TES-like Kinesin 1 (TLK1)) on chromosome 1of Arabidopsis, and a closely homologous rice genomic sequence.Phylogenetic analysis reveals TES, TLK1 and the rice sequence to form astand-alone group, very distinct from other plant kinesins. It isenvisaged that TLK genes can be used in similar ways to TES genes.

[0017] In addition, we have identified a rice homologue of the TES geneand a maize EST that forms part of a maize homologue. It is envisagedthat these rice and maize homologues, and homologues in other plants,will be applicable in the same way as Arabidopsis TES.

[0018] Accordingly, the invention provides:

[0019] a plant in which, by virtue of modulation of the expression of aTETRASPORE (TES) or TES-like (TLK) gene, or of modulation of theactivity of a protein encoded by such a gene, tetrad formation in theanther is disrupted, or is capable of being disrupted, such that callosecross-wall formation in the tetrad fails to the extent that fusionbetween two or more of the four microspore nuclei may occur.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1: TETRASPORE coding region (schematic)

[0021] Stud and tes3 contain a single amino acid subsitution (C-T) andcreate stop codons. tes1, which has a 7 bp deletion, and tes4, whichcontains a 10 bp deletion, alter the reading frame and create stopcodons at 38 bp and 40 bp downstream respectively.

[0022]FIG. 2: RT-PCR expression studies on TES

[0023] TP: four week young plants, R: roots, ST: stems, L: rosetteleaves, YB: young buds before pollen mitosis 1, OB: buds after pollenmitosis 1, FL: open flowers. TES was found to be expressed in all tissuestudies, suggesting that expression is constitutive.

[0024]FIG. 3: Protein line up of TES and homologues

[0025] Line 1 (SEQ ID NO: 1): tes protein=TES, Line 2 (SEQ ID NO: 2):Os=rice homologue, Line 3 (SEQ ID NO: 3): At=TLK1

[0026]FIG. 4: Genomic sequence of wild-type TES in Columbia-3 backgroundand sequence of tes1 mutant

[0027]FIG. 5: Genomic sequence of WS2.R TES and TES-4 mutant

[0028]FIG. 6: Genomic sequence of LER-R TES, and TES-3 and STUD-mutants

DETAILED DESCRIPTION OF THE INVENTION

[0029] Vectors and Chimeric Genes

[0030] Genes and Polynucleotides According to the Invention

[0031] The TES gene whose genomic sequence is provided herein was clonedfrom Arabidopsis. However, the skilled person will appreciate thathomologous genes will exist in other species. Two homologues havealready been identified, namely the rice homologue of SEQ ID No. 2 andthe Arabidopsis TLK1 gene. It will be appreciated that the ricehomologue of SEQ ID No. 2 is a TES or TLK gene as defined herein. Inparticular, it is envisaged that further homologous genes will exist theplants mentioned in the section below entitled “Plants of theinvention”. Based on the genomic DNA sequence and the amino acidsequence of TES provided herein, a skilled person would readily be ableto design probes and primers to identify and obtain homologues in otherplant species. Such homologues form part of the invention.

[0032] A preferred homologue of the invention or polynucleotide of theinvention, which may be isolated form, generally:

[0033] (a) encodes a polypeptide sequence as set out in SEQ ID NO: 1, 2or 3; or

[0034] (b) has a cDNA sequence having 60% or more homology to a sequenceof (a); or

[0035] (c) has a cDNA sequence capable of hybridising selectively to thecomplement of a sequence of (a); and

[0036] when functional, encodes a kinesin protein; and, whennon-functional, causes the plant to fail to form callose cross-walls inthe tetrad.

[0037] Hybridisable Sequences

[0038] A polynucleotide of the invention may hybridise selectively to acoding sequence of (a) at a level significantly above background.Background hybridisation may occur, for example because of other cDNAspresent in a cDNA library. The signal level generated is typically atleast 10 fold, preferably at least 100 fold, as that generated bybackground hybridisation. The intensity of interaction may be measured,for example by radiolabelling the probe, e.g. with ³²P. Selectivehybridisation is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C., for example 45 to 50, 50 to 55 or55 to 60° C., e.g. at 50 or 60° C.

[0039] However, such hybridisation may be carried out under any suitableconditions known in the art (see Sambrook et al, 1989, MolecularCloning: A Laboratory Manual). For example, if high stringency isrequired, suitable conditions include 0.2×SSX at around 60° C., forexample 40 to 50° C., 50 to 60° C. or 60 to 70° C., e.g. at 50 or 60° C.If lower stringency is required, suitable conditions include 2×SSC ataround 60° C., for example 40 to 50° C., 50 to 60° C. or 60 to 70° C.,e.g. at 50 or 60° C.

[0040] Stringency typically occurs in a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the two sequences hybridising toeach other in a duplex) to about 20° C. to 25° C. below Tm. Thus,according to the invention, a hybridisable sequence may be one whichhybridises at a temperature of from Tm to Tm-25° C., e.g. Tm to Tm-5°C., Tm-5 to Tm-10° C., Tm-10 to Tm-20° C. or Tm-20 to Tm-25° C.

[0041] Homologous Sequences

[0042] A polynucleotide sequence of the invention, will typicallycomprise a coding sequence at least 60% or 70%, preferably at least 80or 90% and more preferably at least 95, 98 or 99%, homologous to acoding sequence of (a) above.

[0043] Such homology will preferably apply over a region of at least 20,preferably at least 50, for instance 100 to 500 or more, contiguousnucleotides.

[0044] Methods of measuring nucleic acid and polypeptides homology arewell known in the art. These methods can be applied to measurement ofhomology for both polypeptides and nucleic acids of the invention. Forexample, the UWGCG Package provides the BESTFIT program which can beused to calculate homology (Devereux et al, 1984, Nucleic Acids Research12, p.387-395).

[0045] Similarly, the PILEUP and BLAST algorithms can be used to line upsequences (for example as described in Altschul, S. F., 1993, J Mol.Evol. 30:290-300; Altschul, S. F. et al, 1990) J. Mol. Biol.215:403-410).

[0046] Many different settings are possible for such programs. Accordingto the invention, the default settings may be used.

[0047] In more detail, the BLAST algorithm is suitable for determiningsequence similarity and it is described in Altschul et al (1990) J. Mol.Biol. 215:403-410. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi/nlm.hih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

[0048] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see e.g. Karlin and Altschul (1993)Proc. Natl. Sci. USA 90:5873-5787. One measure of similarity provided bythe BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a fused gene or cDNA if thesmallest sum probability in comparison of the test nucleic acid to afused nucleic acid is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0049] Fragments

[0050] Also included within the scope of the invention are sequenceswhich are fragments of the sequences of (a) to (c) above but have theproperties of the polypeptides of invention as defined above.

[0051] Degenerate Sequences

[0052] Also included within the scope of the invention are sequencesthat differ from those of (b) and (c) above but which, because of thedegeneracy of the genetic code, encode the same polypeptides.

[0053] Complementary Sequences

[0054] In addition, the invention provides polynucleotides havingsequences complementary to any of the above-mentioned sequences. Suchpolynucleotides may be useful in antisense and/or RNAi applications asdiscussed herein.

[0055] Further Properties

[0056] Polynucleotides of the invention may comprise DNA or RNA. Theymay also be polynucleotides which include within them synthetic ormodified nucleotides.

[0057] Polynucleotides of the invention may be used to produce a primer,e.g. a PCR primer, a primer for an alternative amplification reaction, aprobe, e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments willpreferably be at least 10, preferably at least 15 or 20, for example atleast 25, 30 or 40 nucleotides in length. These will be useful inidentifying species homologues and allelic variants as discussed above.

[0058] Polynucleotides such as a DNA polynucleotides and primersaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques. The polynucleotides are typicallyprovided in isolated and/or purified form.

[0059] In general, primers will be produced by synthetic means,involving a stepwise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing this usingautomated techniques are readily available in the art.

[0060] Genomic clones corresponding to the homologues of the inventionand containing, for example introns and promoter regions are alsoaspects of the invention and may also be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques.

[0061] Polynucleotides which are not 100% homologous to the sequences ofthe present invention but fall within the scope of the invention, asdescribed above, can be obtained in a number of ways, for example byprobing cDNA or genomic libraries from other plant species with probesderived from SEQ ID NO: 1, 2, or 3. Degenerate probes can be prepared bymeans known in the art to take into account the possibility ofdegenerate variation between the DNA sequences of SEQ ID NO: 1, 2, or 3and the sequences being probed for under conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.), or other suitable conditions(e.g. as described above).

[0062] Allelic variants and species homologues may also be obtainedusing degenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding likely conserved amino acidsequences. Likely conserved sequences can be predicted from aligning theamino acid sequences of the invention with each other and/or with thoseof any homologous sequences known in the art. The primers will containone or more degenerate positions and will be used at stringencyconditions lower than those used for cloning sequences with singlesequence primers against known sequences.

[0063] Alternatively, such polynucleotides may be obtained bysite-directed mutagenesis. This may be useful where, for example silentcodon changes are required to sequences to optimise codon preferencesfor a particular host cell in which the polynucleotide sequences arebeing expressed. Other sequences may be desired in order to introducerestriction enzyme recognition sites, or to alter the properties orfunction of the polypeptides encoded by the polynucleotides.

[0064] The invention further provides double stranded polynucleotidescomprising a polynucleotide of the invention and its complement.

[0065] Polynucleotides, probes or primers of the invention may carry arevealing label. Suitable labels include radiosotopes such as ³²P or³⁵S, enzyme labels, or other protein labels such as biotin. Such labelsmay be added to polynucleotides, probes or primers of the invention andmay be detected using techniques known per se.

[0066] Polypeptides of the Invention

[0067] Polypeptides of the invention may be encoded by polypeptides asdescribed above. A polypeptide of the invention may consist essentiallyof the amino acid sequence set out in SEQ ID NO: 1 or a substantiallyhomologous sequence, or of a fragment of either of these sequences, aslong as the properties of the invention are maintained.

[0068] In particular, a polypeptide of the invention may comprise:

[0069] (a) the polypeptide sequence of SEQ ID NO: 1, 2 or 3;

[0070] (b) a polypeptide sequence at least 60, 70, 80, 90, 95, 98 or 99%homologous to, a polypeptide of (a); or

[0071] (c) an allelic variant or species homologue of a sequence of (a).

[0072] Allelic Variants

[0073] An allelic variant will be a variant which occurs naturally andwhich will function in a substantially similar manner to the protein ofSEQ ID NO: 1. Similarly, a species homologue of the protein will be theequivalent protein which occurs naturally in another species.

[0074] Homologues

[0075] A polypeptide of the invention is preferably at least 60%homologous to the protein of SEQ ID NO: 1, 2 or 3, more preferably atleast 80 or 90% and more preferably still at least 95, 97 or 99%homologous thereto over a region of at least 20, preferably at least 30,for instance at least 40, 60 or 100 or more contiguous amino acids.Methods of measuring protein homology are well known in the art and itwill be understood by those of skill in the art that in the presentcontext, homology is calculated on the basis of amino acid identity(sometimes referred to as “hard homology”).

[0076] Degrees of homology can be measured by well-known methods, asdiscussed herein for polynucleotide sequences.

[0077] The sequence of the polypeptides of SEQ ID NOs: 1, 2 and 3 and ofthe allelic variants and species homologues can be modified to providefurther polypeptides of the invention.

[0078] Substitutions

[0079] Amino acid substitutions may be made, for example from 1, 2 or 3to 10, 20 or 30 substitutions. For example, a total of up to 1, 2, 5, 10or 20 amino acids may be substituted over a length of 50, 100 or 200amino acids in the polypeptides. For example, up to 20 amino acidssubstituted over any length of 50 amino acids. The modified polypeptidegenerally retains the neurological properties of the invention, asdefined herein. Conservative substitutions may be made, for exampleaccording to the following table. Amino acids in the same block in thesecond column and preferably in the same line in the third column may besubstituted for each other. ALIPHATIC Non-polar G A P I L VPolar-uncharged C S T M N Q Polar-charged D E K R AROMATIC H F W Y

[0080] Fragments

[0081] Polypeptides of the invention also include fragments of theabove-mentioned fall length polypeptides and variants thereof, includingfragments of the sequence set out in SEQ ID NO1: 1, 2 or 3. Suchfragments typically retain the properties of the invention.

[0082] Suitable fragments will generally be at least about 20, e.g. atleast 20, 50 or 100 amino acids in size. Polypeptide fragments of thepolypeptides of SEQ ID NOs: 1, 2 and 3 and allelic and species variantsthereof may contain one or more (e.g. 2, 3, 5, 5 to 10 or more)substitutions, deletions or insertions, including conservativesubstitutions. Each substitution, insertion or deletion may be of anylength, e.g. 1, 2, 3, 4, 5, 5 to 10 or 10 to 20 amino acids in length.

[0083] Isolation and Purification

[0084] Polypeptides and polynucleotides of the invention may be in asubstantially isolated form. It will be understood that they may bemixed with carriers or diluents which will not interfere with theintended purpose of the polypeptide and still be regarded assubstantially isolated. A polypeptide or polynucleotide of the inventionmay also be in a substantially purified form, in which case it willgenerally comprise the polypeptide or polynucleotide, as the case maybe, in a preparation in which more than 70%, e.g. more than 80, 90, 95,98 or 99% of the polypeptide in the preparation is a polypeptide of theinvention.

[0085] Production of Polypeptides

[0086] Polypeptides of the invention may be produced in any suitablemanner. It is preferred that they be produced recombinantly frompolynucleotides of the invention by expression in a suitable host cell,e.g. a bacterial, yeast or plant cell, preferably a plant cell, e.g. ofa species mentioned herein, either in culture or in planta. Polypeptidesmay be recovered and, optionally, purified by techniques known in theart. This can be done using known techniques.

[0087] Replicable Vectors

[0088] Polynucleotides of the invention can be incorporated intorecombinant replicable vectors. Such vectors may be used to replicatethe nucleic acid in a compatible host cell. Thus in a furtherembodiment, the invention provides a method of making polynucleotides ofthe invention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and cultivating the host cell under conditions which bring aboutreplication of the vector. The vector may be recovered from the hostcell. Suitable host cells are described below in connection withexpression vectors. Bacterial cells, especially E. coli are preferred.

[0089] Chimeric Genes and Expression Vectors

[0090] In particular, the invention provides a chimeric gene comprising,operably linked to one or more regulatory sequences capable of securingits expression in a cell, a coding sequence encoding a polypeptide ofthe invention.

[0091] Such chimeric genes can, in turn, be incorporated into expressionvectors. Thus, preferably, a polynucleotide of the invention is operablylinked to regulatory sequences capable of effecting the expression ofthe coding sequence by a host cell. Such chimeric genes and expressionvectors can be used to express the polypeptides of the invention.

[0092] The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is positioned in such a way that expression of thecoding sequence is achieved under conditions compatible with theregulatory sequences.

[0093] Such chimeric genes and expression vectors may be introduced intoa suitable host cell to provide for expression of a polypeptide orpolypeptide fragment of the invention, as described below.

[0094] The vectors may be for example, plasmid, cosmid, virus or phagevectors provided with an origin of replication, preferably a promoterfor the expression of the said polynucleotide and optionally an enhancerand/or a regulator of the promoter. A terminator sequence may also bepresent, as may a polyadenylation sequence. The vectors may contain oneor more selectable marker genes, for example antibiotic ampicillinresistance genes. These will generally be operably linked to regulatorysequences capable of securing their expression in the host cell, asdescribed herein for the coding sequences of the invention.

[0095] Vectors may be used in vitro, for example for the production ofRNA or used to transfect or transform a host cell. The vector may alsobe adapted to be used in vivo, for example for generation of transgenicplants of the invention.

[0096] So far as plasmid vectors are concerned, plasmids derived fromthe Ti plasmid of Agrobacterium tumefaciens are especially preferred, asare plasmids derived from the Ri plasmid of Agrobacterium rhizogens.

[0097] A further embodiment of the invention provides host cellstransformed or transfected with the vectors for the replication andexpression of polynucleotides of the invention. The cells will be chosento be compatible with the said vector and may for example be prokaryotic(bacterial), plant,. yeast, insect or mammalian cells, bacterial andplant cells being preferred.

[0098] Polynucleotides according to the invention may also be insertedinto the vectors described above in an antisense orientation in order toprovide for the production of antisense RNA. Antisense RNA or otherantisense polynucleotides may also be produced by synthetic means. Suchantisense polynucleotides may be used in a method of reducing the levelsof expression of polypeptides having the sequence of SEQ ID NO: 1, orvariants or species homologues thereof in planta.

[0099] An antisense polynucleotide of the invention may be capable ofhybridising to mRNA of a gene of the invention, or a variant or specieshomologue thereof, as defined herein (a “target” mRNA) and may thusinhibit expression by interfering with one or more aspects of mRNAmetabolism including transcription, mRNA processing, mRNA transport fromthe nucleus, translation or mRNA degradation.

[0100] The antisense polynucleotide may be DNA, but is typically RNA.The antisense polynucleotide may be provided as single or doublestranded polynucleotide. The antisense polynucleotide typicallyhybridises to the target mRNA to form a duplex (typically an RNA-RNAduplex) which can cause direct inhibition of translation and/ordestabilisation of the mRNA. Such a duplex may be susceptible todegradation by nucleases.

[0101] The antisense polynucleotide may hybridise to all or part of thetarget mRNA. Typically the antisense polynucleotide hybridises to theribosome binding region or the coding region of the target mRNA. Thepolynucleotide may be complementary to all of or a region of the targetmRNA. For example, the polynucleotide may be the exact complement of allor a part of target mRNA. However, absolute complementary is notrequired and polynucleotides which have sufficient complementarity toform a duplex having a melting temperature of greater than 20° C., 30°C., or 40° C. under physiological conditions are particularly suitablefor use in the present invention. The polynucleotide may be apolynucleotide which hybridises to the target mRNA under conditions ofmedium to high stringency such as 0.03M sodium chloride and 0.03M sodiumcitrate at from about 50° C. to about 60° C.

[0102] In one preferred embodiment the antisense polynucleotide sequenceis complementary to the entire coding sequence of the target mRNA and tothe nucleotides of the mRNA immediately 5′ of the coding sequence.However, the polynucleotide may hybridise to all or part of the 5′- or3′-untranslated region of the mRNA. The antisense polynucleotide may beof any length but will typically be from 6 to 40 nucleotides in length.More preferably it will be from 12 to 20 nucleotides in length. Thepolynucleotide may be at least 40, for example at least 60 or at least80, nucleotides in length and up to 100, 200, 300, 400, 500, 1000 ormore nucleotides in length. In one embodiment the length of theantisense oligonucleotide is the same as that of the target mRNA or upto a few nucleotides, such as 5 or 10 nucleotides, shorter than themRNA.

[0103] Promoters and other regulatory elements may be selected to becompatible with the host cell for which the expression vector isdesigned.

[0104] Promoters suitable for use in plant cells may be derived, forexample, from plants or from bacteria that associate with plants or fromplant viruses. Thus, promoters from Agrobacterium spp. including thenopaline synthase (nos), octopine synthase (ocs) and mannopine synthase(mas) promoters are preferred. Also preferred are plant promoters suchas the ribulose bisphosphate small subunit promoter (rubisco ssu),histone promoters (EP-A-0 507,698), the rice actin promoter (U.S. Pat.No. 5,641,876) and the phaseolin promoter. Also preferred are plantviral promoters such as the cauliflower mosaic virus (CAMV) 35S and 19Spromoters, and the circovirus promoter (AU-A-689,311).

[0105] Depending on the pattern of expression desired, promoters may beconstitutive, tissue- or stage-specific; and/or inducible. For example,strong constitutive expression in plants can be obtained with the CAMV35S, Rubisco ssu, or histone promoters mentioned above. Also,tissue-specific or stage-specific promoters may be used to targetexpression of polypeptides of the invention to particular tissues in atransgenic plant or to particular stages in its development. Promotersspecific to the anther, or to early anther development are particularlyadvantageous.

[0106] Inducible promoters are particularly preferred. Alcohol-inducibleand herbicide-inducible promoters are available. Chemically induciblepromoters such as those activated by herbicide safeners may also beused, for example the maize GST 27 promoter (WO97/11189), the maizeIn2-1 promoter (WO90/11361), the maize In2-2 promoter (De Veylder et al,Plant Cell Physiology, Vol.38, pp568-577 (1997).

[0107] Especially where expression in plant cells is desired, otherregulatory signals may also be incorporated in the vector, for example aterminator and/or polyadenylation site. Preferred terminators includethe nos terminator and the histone terminator of EP-A-0 633,317 althoughother terminators functional in plant cells may also be used.Additionally, sequences encoding secretory signals or transit peptidesmay be included. On expression, these elements direct secretion from thecell or target the polypeptide of the invention to a particular locationwithin the cell. For example, sequences may be added to target theexpressed polypeptide to the nucleus or plastids (e.g. chloroplasts) ofa plant cell.

[0108] Some examples are signal-peptide encoding DNA/RNA sequences whichtarget proteins to the extracellular matrix of the plant cell, such asthe signal sequence of the Nicotiana plumbaginifolia extension gene;signal peptides which target proteins to the vacuole, like those of thesweet potato sporamin gene and the barley lectin gene; signal peptideswhich cause proteins to be secreted such as that of PRIb; or the barleyα-amylase leader sequence; and signal peptides which target proteins tothe plastids such as that of rapeseed enoyl-Acp reductase.

[0109] Typically, therefore, a chimeric gene comprises the followingelements in 5′ to 3′ orientation: a promoter functional in a host(preferably plant) cell, as defined above, a polynucleotide of theinvention and a terminator functional in said cell, as defined above.

[0110] Other elements, for example enhancers, may also be present in avector of the invention. Enhancers include the tobacco etch virus (TEV)enhancer and the tobacco. mosaic virus (TMV) enhancer (WO87/07644).

[0111] Similarly, an origin of replication may be present. Sequencescapable of securing integration into a cells genome, e.g. Agrobacteriumtumefaciens T-DNA sequences may be present.

[0112] Further, selectable marker genes, under control of their ownregulatory sequences may be included. These include antibioticresistance genes. Examples include genes that confer resistance to theantibiotics kanamycin and/or neomycin (e.g. the nptI and nptII genes) orchloramphenicol (e.g. the CAT gene). Herbicide resistance genes may alsobe used as selectable markers. Notably, genes conferring resistance toherbicides such as bialaphos, glyphosate or an isoxazole herbicide maybe used. Particular examples are described in EP-A-0 242,236, EP-A-0242,246, GB-A-2,197,653, WO91/02701, WO95/06128, WO96/38567 andWO97/04103. Likewise, scorable marker genes may be present. Someexamples are the β-glucuronidase (GUS) β-galactosidase luciferase andgreen fluorescent protein (gfp) genes.

[0113] Expression in Host Cells

[0114] Expression in the host cell may be transient although,preferably, integration of the polynucleotide or chimeric gene of theinvention into the cell's genome is achieved.

[0115] Cell culture will take place under standard conditions.Commercially available cultural media for cell culture are widelyavailable and can be used in accordance with manufacturers'instructions.

[0116] Plants of the Invention

[0117] The present invention is in principle applicable to any plantspecies, notably arable crop species, tree species and species used inhorticulture, especially ornamentals. Preferred dicotyledonous cropplants include tomato; potato; sugarbeet cassava; cruciferous crops,including oilseed rape; linseed; tobacco; sunflower; fibre crops such ascotton; and leguminous crops such as peas, beans, especially soybean,and alfalfa. Brassicas are particularly preferred. Preferredmonocotyledonous plants include graminaceous plants such as wheat,maize, rice, oats, barley, rye, sorghum, triticale and sugar cane. Maizeis particularly preferred.

[0118] The invention is also particularly useful in the context oftransgenic trees, where male sterility is of benefit because it preventsdispersal of transgenic pollen.

[0119] Transformation/Regeneration Techniques

[0120] The cell used for transformation may be from any suitableorganism, preferably a plant as defined herein and may be in any form.For example, it may be an isolated cell, e.g. a protoplast, or it may bepart of a plant tissue, e.g. a callus, for example a solid or liquidcallus culture, or a tissue excised from a plant, or it may be part of awhole plant. It may, for example, be part of an embryo, or a meristem,e.g. an apical meristem of a shoot. Transformation may thus give rise toa chimeric tissue or plant in which some cells are transgenic and someare not.

[0121] Transformation Techniques

[0122] Cell transformation may be achieved by any suitabletransformation method, for example the transformation techniquesdescribed herein. Preferred transformation techniques includeelectroporation of plant protoplasts (Taylor and Walbot, 1985),PEG-based procedures (Golds et al, 1993), microinjection (Neuhas et al,1987; Potrykus et al, 1985), injection by galinstan expansionfemtosyringe (Knoblauch et al, 1999), Agrobacterium-mediatedtransformation and particle bombardment. Particle bombardment isparticularly preferred.

[0123] Selection of Transformed Cells

[0124] Cells generated by the transformation techniques discussed abovewill typically be present in chimeric tissues, and thus will besurrounded by other non-transformed cells. Standard selection techniquesusing co-transforming selectable and/or scorible markers can then beused to identify and obtain transformed cells.

[0125] Regeneration and Breeding

[0126] Transformed cells may be regenerated into a transgenic plant bytechniques known in the art. These may involve the use of plant growthsubstances such as auxins, giberellins and/or cytokinins to stimulatethe growth and/or division of the transgenic cell. Similarly, techniquessuch as somatic embryogenesis and meristem culture may be used.Regeneration techniques are well known in the art and examples can befound in, e.g. U.S. Pat. No. 4,459,355, U.S. Pat. No. 4,536,475, U.S.Pat. No. 5,464,763, U.S. Pat No. 5, 177,010, U.S. Pat. No. 5,187,073, EP267,159, EP 604, 662, EP 672, 752, U.S. Pat. No. 4,945,050, U.S. Pat No.5,036,006, U.S. Pat. No. 5,100,792, U.S. Pat. No. 5,371,014, U.S. PatNo. 5,478,744, U.S. Pat. No 5,179,022, U.S. Pat. No. 5,565,346, U.S.Pat. No. 5,484,956, U.S. Pat. No. 5,508,468, U.S. Pat. No. 5,538,877,U.S. Pat. No. 5,554,798, U.S. Pat. No. 5,489,520, U.S. Pat. No.5,510,318, U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,405,765, EP 442,174,EP 486,233, EP 486,234, EP 539,563, EP 674,725, WO91/02071, WO 95/06128and WO 95/32977.

[0127] In many such techniques, one step is the formation of a callus,i.e. a plant tissue comprising expanding and/or dividing cells. Suchcalli are a further aspect of the invention as are other types of plantcell cultures and plant parts. Thus, for example, the invention providestransgenic plant tissues and parts, including embryos, meristems, seeds,shoots, roots, stems, leaves and flower parts. These may be chimeric inthe sense that some of their cells are transgenic and some are not.

[0128] Regeneration procedures will typically involve the selection oftransgenic cells by means of marker genes. The regeneration step givesrise to a first generation transgenic plant. The invention also providesmethods of obtaining transgenic plants of further generations from thisfirst generation plant. These are known as progeny plants. Progenyplants of second, third, fourth, fifth, sixth and further generationsmay be obtained from the first generation progeny plant by any meansknown in the art.

[0129] Thus, the invention provides a method of obtaining a transgenicplant of the invention comprising obtaining a second-generationtransgenic progeny plant from a first-generation progeny plant of theinvention, and optionally obtaining transgenic plants of one or morefurther generations from the second-generation progeny plant thusobtained.

[0130] Such progeny plants are desirable because the first generationplant may not have all the characteristics required for cultivation. Forexample, for the production of first generation transgenic plants, aplant of a taxon that is easy to transform and regenerate may be chosen.It may therefore be necessary to introduce further characteristics inone or more subsequent generations of progeny plants before a plant moresuitable for cultivation is produced.

[0131] Progeny plants may be produced from their predecessors of earliergenerations by any known technique. In particular, progeny plants may beproduced by:

[0132] obtaining a transgenic seed from a transgenic plant of theinvention belonging to a previous generation, then obtaining atransgenic progeny plant of the invention belonging to a new generationby growing up the seed; and/or

[0133] propagating clonally a transgenic plant of the inventionbelonging to a previous generation to give a transgenic progeny plant ofthe invention belonging to a new generation; and/or

[0134] crossing a first-generation transgenic plant of the inventionbelonging to a previous generation with another compatible plant to givea transgenic progeny plant of the invention belonging to a newgeneration; and optionally

[0135] obtaining transgenic progeny plants of one or more furthergenerations from the progeny plant thus obtained.

[0136] These techniques may be used in any combination. For example,clonal propagation and sexual propagation may be used at differentpoints in a process that gives rise to a plant suitable for cultivation.In particular, repetitive back-crossing with a plant taxon withagronomically desirable characteristics may be undertaken. Further stepsof removing cells from a plant and regenerating new plants therefrom mayalso be carried out.

[0137] Also, further desirable characteristics may be introduced bytransforming the cells, plant tissues, plants, or seeds, at any suitablestage in the above process, to introduce desirable coding sequencesother than the polynucleotides of the invention. This may be carried outby conventional breeding techniques, e.g. fertilizing a plant of theinvention with pollen from a plant with the desired additionalcharacteristic. Alternatively, the characteristic can be added byfurther transformation of the plant obtained by the method of theinvention. Preferably, different transgenes are linked to differentselectable of scorable markers to allow selection for both the presenceof further transgenes. Selection, regeneration and breeding techniquesfor nuclear transformed plants are known in the art.

[0138] Uses of Plants of the Invention

[0139] The invention also provides methods of obtaining crop products byharvesting, and optionally processing further, cells, calli, plants orseeds of the invention. By crop product is meant any useful productobtainable from a crop plant.

[0140] Such a product may be obtainable directly by harvesting orindirectly, by harvesting and further processing. Directly obtainableproducts include: grains, e.g. grains of monocotyledonous species,preferably graminaceous species, for example wheat, oats, rye, rice,maize, sorghum, triticale, especially wheat; other seeds; shoots,especially tubers, such as potato tubers; fruit; and other plant parts,for example as defined herein. Alternatively, such a product may beobtainable indirectly, by harvesting and further processing. Examples ofproducts obtainable by further processing are: flour; oil; rubber;beverages such as juices and fermented and/or distilled alcoholicbeverages; food products made from directly obtained or furtherprocessed material, e.g. bread made from flour or margarine made fromoil; tobacco and tobacco products such as cigarettes and cigars; fibres,e.g. cotton, linen, flax and hemp fibres and textile items madetherefrom; paper or timber derived from woody plants.

[0141] Additional Features of Plants of the Invention

[0142] In addition to the transgenes of the invention, plants of theinvention may be transgenic in other respects. For example, they may betransformed such that they comprise genes for herbicide, insecticide ordisease resistance. Preferred herbicide resistance genes may beresponsible for, for example, tolerance to: Glyphosate (e.g. using anEPSP synthase gene (e.g. EP-A-0 293,358) or a glyphosate oxidoreductase(WO 92/000377) gene); or tolerance to fosametin; a dihalobenzonitrile;glufosinate, e.g. using a phosphinothrycin acetyl transferase (PAT) orglutamine synthase gene (cf. EP-A-0 242,236); asulam, e.g. using adihydropteroate synthase gene (EP-A-0 369,367); or a sulphonylurea, e.g.using an ALS gene); diphenyl ethers such as acifluorfen or oxyfluorfen,e.g. using a protoporphyrogen oxidase gene); an oxadiazole such asoxadiazon; a cyclic imide such as chlorophthalim; a phenyl pyrazole suchas TNP, or a phenopylate or carbamate analogue thereof; spectinomycine.g. using the aadA gene, as exemplified below.

[0143] Insect resistance may be introduced, for example using genesencoding Bacillus thuringiensis (Bt) toxins. Likewise, genes for diseaseresistance may be introduced, e.g. as in WO91/02701 or WO95/06128.

[0144] Transformation may also lead to the introduction of a selectablemarker gene i.e. marker genes that allow transformed cells to survive inthe presence of agents that kill non-transformed cells. Any selectablemarker gene may be used in the transforming polynucleotide of theinvention. Some examples have already been given above. Typically,herbicide resistance genes, e.g. as defined above, may be used asselectable markers. Alternatively, coding regions that encode productswhich provide resistance to aminoglycoside antibiotics may be used asselectable marker, for example, encoded products that provide resistanceto kanomycin, neomycin or chloramphenicol. The encoded polypeptide maycause morphological alterations to cultured transformed cells, such asisopentyltransferase (Kunkel et al, 1999). The encoded polypeptide maybe a scorable marker, which allows transformed cells to be distinguishedfrom non-transformed cells, generally by alteration of the transformedcell's optical properties. Any scorable marker may be used. Preferredscorable markers include, polypeptides which are able to alter theappearance or optical properties of transformed cells, for example:β-glucoronidase (i.e. the uidA:GUS gene); fluorescent proteins such asgreen fluorescent protein (GFP), yellow fluorescent protein (YFP) orcyan fluorescent protein (CFP); or luminescent proteins such asluciferase or aequorin. Cells with scorable optical differences can besorted using techniques such as fluorescence activated cell sorting(FACS). In a preferred embodiment, the polynucleotide of the inventioncomprises a selectable marker and a scorable marker, for example, theFLARE-S marker genes which comprise aadA and GFP (Khan and Maliga,1999).

[0145] Similarly, plants of the invention may be transformed such thatthey express polypeptides whose mass production is desirable, e.g.components of antibodies, or pharmaceutically active polypeptides suchas interferon-gamma.

[0146] Strategies for Exploitation of TES and TLK Genes

[0147] Desirably, down-regulation of TES and/or TLK genes according tothe invention will be inducible, eg by means of the techniques describedherein. Constitutive down-regulation is less desirable as it will leadto the production of partially fertile plants. The key stage fordown-regulation of TES and/or TLK is at the point of pollen productionand fertilisation.

[0148] Another preferred technique is therefore use ofrecombinase-encoding constructs to excise, for example, antisense orRNAi constructs according to the invention. Recombinase systems to dothis are available in the art. In outline, a construct, for example, anantisense construct of the invention, is flanked by sequences that arerecognised by the recombinase enzyme. The recombinase enzyme is thencapable of splicing the flanking sequences together, thus excising theconstruct of the invention. In a particularly preferred embodiment,down-regulation of TES and/or TLK achieved by means of an introducedantisense or RNAi construct. Of course, this plant is generally the maleparent plant in any cross. The female parent plant in the cross istransformed with a construct encoding a recombinase enzyme, and theantisense or RNAi construct in the male parent plant is flanked by thesequences recognised by the recombinase. Thus, when the two plants arecrossed, the recombinase is expressed in the progeny and splices out theantisense or RNAi construct. The use of recombinases in this manner isparticularly advantageous because it eliminates the transgenic constructof the invention, which is beneficial from the point of view of publicperception. Even more desirably, the recombinase construct may beself-excising, in that it is itself flanked by sequences which theenzyme it encodes can excise. Alternatively, the transgene can besegregated out by conventional techniques.

[0149] TES and TLK genes also have implications in increasing seed size.This is particularly desirable in graminaceous crops where it is theseed that is the part of the plant that is consumed by humans andanimals. Increased seed size is desirable because it may increaseoverall yield and, even if it does not, it may increase seed size at theexpense of the number of seeds. This is advantageous because it reducesthe amount of processing required. It is envisaged that overexpressionof TES and/or TLK genes will increase seed size.

[0150] Antibodies

[0151] The invention also provides monoclonal or polyclonal antibodieswhich specifically recognise polypeptides of the invention, and methodsof making such antibodies. Antibodies of the invention bind specificallyto the polypeptides of the invention.

[0152] Monoclonal antibodies may be prepared by conventional hybridomatechnology using polypeptides of the invention as immunogens. Polyclonalantibodies may also be prepared by conventional means which compriseinoculating a host animal, for example a rat or a rabbit, with apolypeptide of the invention, or a fragment thereof comprising anepitope, and recovering immune serum. In order that such antibodies maybe made, polypeptides may be haptenised to another polypeptide for useas immunogens in animals or humans. For the purposes of this invention,the term “antibody” includes antibody fragments such as Fv, F(ab) andF(ab)₂ fragments, as well as single-chain antibodies.

[0153] Antibodies to the polypeptides of the invention can be producedby use of the following methods. An antibody to the substance may beproduced by raising antibody in a host animal against the wholesubstance or an antigenic epitope thereof (hereinafter “the immunogen”).Methods of producing monoclonal and polyclonal antibodies arewell-known.

[0154] A method for producing a polyclonal antibody comprises immunisinga suitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the serum. The animal maytherefore be inoculated with the immunogen, blood subsequently removedfrom the animal and the IgG faction purified.

[0155] A method for producing a monoclonal antibody comprisesimmortalising cells which produce the desired antibody. Hybridoma cellsmay be produced by fusing spleen cells from an inoculated experimentalanimal with tumour cells (Kohler and Milstein, Nature (1975) 256,495-497).

[0156] An immortalised cell producing the desired antibody may beselected by a conventional procedure. The hybridomas may be grown inculture or injected intraperitoneally for formation of ascites fluid orinto the blood stream of an allogenic host or immunocompromised host.Human antibody may be prepared by in vitro immunisation of humanlymphocytes, followed by transformation of the lymphocytes withEpstein-Barr virus.

[0157] For production of both monoclonal and polyclonal antibodies, theexperimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled, for example via a side chain of one of the aminoacid residues, to a suitable carrier. The carrier molecule is typicallya physiologically acceptable carrier. The antibody obtained may beisolated and, if desired, purified.

[0158] Thus, the invention provides the use of a polypeptide of theinvention, or a fragment thereof comprising an epitope, in theproduction of antibodies that specifically recognise a polypeptide ofthe invention. For these purposes, it should be noted that the fragmentsmay not function as plant-protective polypeptides, because an epitopemay be contained within a region too small to retain function as aplant-protective polypeptide. Similarly, the invention provides methodsof producing antibodies by inoculating animals with a polypeptide of theinvention or a fragment thereof containing an epitope and recoveringimmune serum. This will generate polyclonal antibodies.

[0159] Antibodies may also be generated using β-cells in vitro insteadof in vivo.

[0160] Antibodies to polypeptides of the invention may also beidentified by phage display techniques.

[0161] Antibodies of the invention can be used to identify compoundswhose structural properties (e.g. shape, charge) correspond to those ofthe polypeptides of the invention. Thus, they may be used to screen forcompounds that mimic the functional properties of the polypeptides ofthe invention.

[0162] The invention is illustrated below by means of the followingExamples.

EXAMPLES

[0163] 1. Screening Recombinants of tes Individuals in F2 MappingPopulation

[0164] The tes1 mutant (Columbia background) (Spielman et al (1997),supra) was crossed with wild-type Ler. F1 plants were selfed andphenotypically tes individuals in F2 were scored and genoinic DNA fromeach individual was extracted.

[0165] A total of 891 phenotypically tes individuals were obtained.Molecular markers ATPOX/T20P21 (CAPs), north of tes, and NIT1 (SSLP),1cM south of tes were used to screen the mapping population. With markerATPOX, 13 out of 891 phenotypically tes individuals were recombinants,51 recombinants with NIT1. Primers were generated according to sequencedBAC ends within the region between ATPOX and NIT1 and used to score therecombinants from both sides and ‘walk’ towards the tes locus.

[0166] With primers from one end of BAC clone F7K15, 5 recombinants frommarker ATPOX side were detected. BAC clone T5C2 has 10 kb sequenceoverlap with the other end of F7K15. One recombinant was observed byusing primers from T5C2 end on NIT1 side. No recombinants were detectedwith primers from the region between the two overlapped clones.

[0167] 2. Detection of Polymorphisms Between tes Alleles and Wild TypePlants (Ecotypes)

[0168] TES has been mapped to two overlapped Arabidopsis BAC clones(F7K15 and T5C2) which have been sequenced and annotated. The size ofthe F7K15 clone is 110800 bp containing 15 predicted proteins and 4transposons. T5C2 is 103098 bp having 13 predicted proteins and 7transposons.

[0169] (1) Southern blot analysis has been done on three ecotypes (Col,Ler and WS2) and corresponding tes alleles using the two BAC clones asprobes to detect RFLPs associated with mutant alleles. No polymorphismswere observed with 9-20 restriction enzyme digested DNA blots.

[0170] (2) PCR based analysis (CAPs) of tes alleles and wild-type plantswas conducted using primers corresponding to the sequences of F7K15 andT5C2 clones. The size of the PCR products were from 5 kb to 10 kb andthey were overlapped from 0.5 kb to 1 kb. PCR products digested withrestriction enzymes were screened for polymorphisms. Polymorphisms wereobserved within a predicted kinesin-like protein region (F7K15-60 from27071 to 31660 bp on F7K15 BAC sequence) with restriction enzymes Alu1,Dde1 and Sau3al on alleles tes1 and tes4 (Spielman et al (1997), supra).The experimental detection of polymorphisms has been repeated andconfirmed by several independent experiments.

[0171] tes1 was mutated on ecotype Col-3, while sequence data obtainedfrom ecotype Col-0. Col-3 and Col-0 showed the same restrictiondigestion pattern on the tes region. This confirmed that tes1 mutationsare not due to ecotype background.

[0172] 3. Sequence Analysis of tes Region

[0173] The size of the tes genomic sequence is 4589 bp encoding 932amino acids. Both strands of ˜7.5 kb region from 4 alleles and 3ecotypes have been sequenced. Results showed that tes4 (T-DNAmutagenesis) has a 10 bp deletion, which alters the reading frame. Astop code is created at 40 bp downstream of the deletion; tes3 and stud(EMS mutagenesis) have one base pair substitutions (C-T) and place stopcodons (CAA-TAA; CGA-TGA) in the kinesin motor domain. tes1 (fastneutron mutagenesis) contains 7 bp deletion causing a Frameshift andcreating a stop code 38 bp downstream. Mutations of tes4, tes3 and studare located in the third last exon of 14 exons, the tes1 mutation is inthe last exon of the gene.

[0174] Database searches have identified a very similar protein to TESon chromosome 1 of Arabidopsis and a homologous rice kinesin sequence(see FIG. 3). Phylogenetic analysis reveals TES and the two homologouskinesin forming a distinct group.

[0175] 4. Expression Analysis of tes

[0176] RT-PCR expression analysis showed TES to be expressed in allparts of the Arabidopsis plant (FIG. 2).

1 10 1 932 PRT Arabidopsis thaliana 1 Met Gly Pro Pro Arg Thr Pro LeuSer Lys Ile Asp Lys Ser Asn Pro 1 5 10 15 Tyr Thr Pro Cys Gly Ser LysVal Thr Glu Glu Lys Ile Leu Val Thr 20 25 30 Val Arg Met Arg Pro Leu AsnTrp Arg Glu His Ala Lys Tyr Asp Leu 35 40 45 Ile Ala Trp Glu Cys Pro AspAsp Glu Thr Ile Val Phe Lys Asn Pro 50 55 60 Asn Pro Asp Lys Ala Pro ThrLys Tyr Ser Phe Asp Lys Val Phe Glu 65 70 75 80 Pro Thr Cys Ala Thr GlnGlu Val Tyr Glu Gly Gly Ser Arg Asp Val 85 90 95 Ala Leu Ser Ala Leu AlaGly Thr Asn Ala Thr Ile Phe Ala Tyr Gly 100 105 110 Gln Thr Ser Ser GlyLys Thr Phe Thr Met Arg Gly Val Thr Glu Ser 115 120 125 Val Val Lys AspIle Tyr Glu His Ile Arg Lys Thr Gln Glu Arg Ser 130 135 140 Phe Val LeuLys Val Ser Ala Leu Glu Ile Tyr Asn Glu Thr Val Val 145 150 155 160 AspLeu Leu Asn Arg Asp Thr Gly Pro Leu Arg Leu Leu Asp Asp Pro 165 170 175Glu Lys Gly Thr Ile Val Glu Asn Leu Val Glu Glu Val Val Glu Ser 180 185190 Arg Gln His Leu Gln His Leu Ile Ser Ile Cys Glu Asp Gln Arg Gln 195200 205 Val Gly Glu Thr Ala Leu Asn Asp Lys Ser Ser Arg Ser His Gln Ile210 215 220 Ile Arg Leu Thr Ile His Ser Ser Leu Arg Glu Ile Ala Gly CysVal 225 230 235 240 Gln Ser Phe Met Ala Thr Leu Asn Leu Val Asp Leu AlaGly Ser Glu 245 250 255 Arg Ala Phe Gln Thr Asn Ala Asp Gly Leu Arg LeuLys Glu Gly Ser 260 265 270 His Ile Asn Arg Ser Leu Leu Thr Leu Thr ThrVal Ile Arg Lys Leu 275 280 285 Ser Ser Gly Arg Lys Arg Asp His Val ProTyr Arg Asp Ser Lys Leu 290 295 300 Thr Arg Ile Leu Gln Asn Ser Leu GlyGly Asn Ala Arg Thr Ala Ile 305 310 315 320 Ile Cys Thr Ile Ser Pro AlaLeu Ser His Val Glu Gln Thr Lys Lys 325 330 335 Thr Leu Ser Phe Ala MetSer Ala Lys Glu Val Thr Asn Cys Ala Lys 340 345 350 Val Asn Met Val ValSer Glu Lys Lys Leu Leu Lys His Leu Gln Gln 355 360 365 Lys Val Ala LysLeu Glu Ser Glu Leu Arg Ser Pro Glu Pro Ser Ser 370 375 380 Ser Thr CysLeu Lys Ser Leu Leu Ile Glu Lys Glu Met Lys Ile Gln 385 390 395 400 GlnMet Glu Ser Glu Met Lys Glu Leu Lys Arg Gln Arg Asp Ile Ala 405 410 415Gln Ser Glu Leu Asp Leu Glu Arg Lys Ala Lys Glu Arg Lys Gly Ser 420 425430 Ser Glu Cys Glu Pro Phe Ser Gln Val Ala Arg Cys Leu Ser Tyr His 435440 445 Thr Lys Glu Glu Ser Ile Pro Ser Lys Ser Val Pro Ser Ser Arg Arg450 455 460 Thr Ala Arg Asp Arg Arg Lys Asp Asn Val Arg Gln Ser Leu ThrSer 465 470 475 480 Ala Asp Pro Thr Ala Leu Val Gln Glu Ile Arg Leu LeuGlu Lys His 485 490 495 Gln Lys Lys Leu Gly Glu Glu Ala Asn Gln Ala LeuAsp Leu Ile His 500 505 510 Lys Glu Val Thr Ser His Lys Leu Gly Asp GlnGln Ala Ala Glu Lys 515 520 525 Val Ala Lys Met Leu Ser Glu Ile Arg AspMet Gln Lys Ser Asn Leu 530 535 540 Leu Thr Glu Glu Ile Val Val Gly AspLys Ala Asn Leu Lys Glu Glu 545 550 555 560 Ile Asn Arg Leu Asn Ser GlnGlu Ile Ala Ala Leu Glu Lys Lys Leu 565 570 575 Glu Cys Val Gln Asn ThrIle Asp Met Leu Val Ser Ser Phe Gln Thr 580 585 590 Asp Glu Gln Thr ProAsp Phe Arg Thr Gln Val Lys Lys Lys Arg Leu 595 600 605 Leu Pro Phe GlyLeu Ser Asn Ser Pro Asn Leu Gln His Met Ile Arg 610 615 620 Gly Pro CysSer Pro Leu Ser Gly Thr Glu Asn Lys Asp Pro Glu Ser 625 630 635 640 AsnVal Val Ser Ala Asn Ser Ala Pro Val Ser Phe Gly Ala Thr Pro 645 650 655Pro Lys Arg Asp Asp Asn Arg Cys Arg Thr Gln Ser Arg Glu Gly Thr 660 665670 Pro Val Ser Arg Gln Ala Asn Ser Val Asp Ile Lys Arg Met Asn Arg 675680 685 Met Tyr Lys Asn Ala Ala Glu Glu Asn Ile Arg Asn Ile Lys Ser Tyr690 695 700 Val Thr Gly Leu Lys Glu Arg Val Ala Lys Leu Gln Tyr Gln LysGln 705 710 715 720 Leu Leu Val Cys Gln Ala Asn Glu Thr Gly Ala Ala SerGlu Tyr Asp 725 730 735 Ala Thr Asp Glu Ser Gln Met Asp Trp Pro Leu CysPhe Glu Glu Gln 740 745 750 Arg Lys Gln Ile Ile Met Leu Trp His Leu CysHis Ile Ser Ile Ile 755 760 765 His Arg Thr Gln Phe Tyr Met Leu Phe LysGly Asp Pro Ala Asp Gln 770 775 780 Ile Tyr Met Glu Val Glu Leu Arg ArgLeu Thr Trp Leu Glu Gln His 785 790 795 800 Leu Ala Glu Leu Gly Asn AlaSer Pro Ala Leu Leu Gly Asp Glu Pro 805 810 815 Ala Ser Tyr Val Ala SerSer Ile Arg Ala Leu Lys Gln Glu Arg Glu 820 825 830 Tyr Leu Ala Lys ArgVal Asn Thr Lys Leu Gly Ala Glu Glu Arg Glu 835 840 845 Met Leu Tyr LeuLys Trp Asp Val Pro Pro Val Gly Lys Gln Arg Arg 850 855 860 Gln Gln PheIle Asn Lys Leu Trp Thr Asp Pro His Asn Met Gln His 865 870 875 880 ValArg Glu Ser Ala Glu Ile Val Ala Lys Leu Val Gly Phe Cys Asp 885 890 895Ser Gly Glu Thr Ile Arg Lys Glu Met Phe Glu Leu Asn Phe Ala Ser 900 905910 Pro Ser Asp Lys Lys Thr Trp Met Met Gly Trp Asn Phe Ile Ser Asn 915920 925 Leu Leu His Leu 930 2 954 PRT Oryza sativa 2 Met Gly Val Ser ArgPro Pro Ser Thr Pro Ala Ser Lys Ile Glu Arg 1 5 10 15 Thr Pro Met SerThr Pro Thr Pro Gly Gly Ser Thr Arg Val Lys Glu 20 25 30 Glu Lys Ile PheVal Thr Val Arg Val Arg Pro Leu Ser Lys Lys Glu 35 40 45 Leu Ala Leu LysAsp Gln Val Ala Trp Glu Cys Asp Asp Asn Gln Thr 50 55 60 Ile Leu Tyr LysGly Pro Pro Gln Asp Arg Ala Ala Pro Thr Ser Tyr 65 70 75 80 Thr Phe AspLys Val Phe Gly Pro Ala Ser Gln Thr Glu Val Val Tyr 85 90 95 Glu Glu GlyAla Lys Asp Val Ala Met Ser Ala Leu Thr Gly Ile Asn 100 105 110 Ala ThrIle Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr Phe Thr 115 120 125 MetArg Gly Val Thr Glu Ser Ala Val Asn Asp Ile Tyr Arg His Ile 130 135 140Glu Asn Thr Pro Glu Arg Asp Phe Ile Ile Lys Ile Ser Ala Met Glu 145 150155 160 Ile Tyr Asn Glu Ile Val Lys Asp Leu Leu Arg Pro Glu Ser Thr Asn165 170 175 Leu Arg Leu Leu Asp Asp Pro Glu Lys Gly Thr Ile Val Glu LysLeu 180 185 190 Glu Glu Glu Ile Ala Lys Asp Ser Gln His Leu Arg His LeuIle Ser 195 200 205 Ile Cys Glu Glu Gln Arg Gln Val Gly Glu Thr Ala LeuAsn Asp Thr 210 215 220 Ser Ser Arg Ser His Gln Ile Ile Arg Leu Thr ValGlu Ser Arg Leu 225 230 235 240 Arg Glu Val Ser Gly Cys Val Lys Ser PheVal Ala Asn Leu Asn Phe 245 250 255 Val Asp Leu Ala Gly Ser Glu Arg AlaAla Gln Thr His Ala Val Gly 260 265 270 Ala Arg Leu Lys Glu Gly Cys HisIle Asn Arg Ser Leu Leu Thr Leu 275 280 285 Thr Thr Val Ile Arg Lys LeuSer Ser Asp Lys Arg Ser Gly His Ile 290 295 300 Pro Tyr Arg Asp Ser LysLeu Thr Arg Ile Leu Gln Leu Ser Leu Gly 305 310 315 320 Gly Asn Ala ArgThr Ala Ile Ile Cys Thr Met Ser Pro Ala Gln Thr 325 330 335 His Val GluGln Ser Arg Asn Thr Leu Phe Phe Ala Thr Cys Ala Lys 340 345 350 Glu ValThr Asn Asn Ala Lys Val Asn Met Val Val Ser Asp Lys Gln 355 360 365 LeuVal Lys His Leu Gln Met Glu Val Ala Arg Leu Glu Ala Glu Leu 370 375 380Arg Thr Pro Asp Arg Ala Ser Ser Ser Glu Ile Ile Ile Met Glu Arg 385 390395 400 Asp Arg Lys Ile Arg Gln Met Glu Lys Glu Met Glu Glu Leu Lys Lys405 410 415 Gln Arg Asp Asn Ala Gln Leu Lys Leu Glu Glu Leu Gln Lys LysMet 420 425 430 Gly Asp Asn Gln Pro Gly Trp Asn Pro Phe Asp Ser Pro GlnArg Thr 435 440 445 Arg Lys Cys Leu Thr Tyr Ser Gly Ser Leu Gln Pro SerAsn Lys Met 450 455 460 Lys Ile Arg Ser Ser Ile Arg Gln Ser Ala Thr AlaPro Phe Met Leu 465 470 475 480 Lys His Glu Ile Arg Lys Leu Glu Gln LeuGln Gln Gln Leu Glu Val 485 490 495 Glu Ala Asn Arg Ala Ile Glu Val LeuHis Lys Glu Val Glu Cys His 500 505 510 Lys His Gly Asn Gln Asp Ala AlaGlu Thr Ile Ala Lys Leu Gln Ala 515 520 525 Glu Ile Arg Gly Met Gln SerVal Arg Ser Asp Arg Asp Val Asp Met 530 535 540 Ile Thr Asp Glu Gly AsnGly Ser Asp Leu Lys Glu Glu Ile Ser Arg 545 550 555 560 Leu His Met GlnAsp Asn Asp Ile Ala Lys Leu Glu Ala Lys Leu Glu 565 570 575 Asn Val GlnArg Ser Ile Asp Arg Leu Val Met Ser Leu Pro Asn Val 580 585 590 Gly ThrGln Cys Asn Glu Thr Thr Pro Lys Ser Asn Arg Ala Lys Lys 595 600 605 LysLys Arg Met Leu Leu Pro Leu Gly Val Ser Asn Ile Asn Arg Pro 610 615 620Asn Leu Ile Arg Ala Pro Cys Ser Pro Leu Ser Ser Ser Arg Pro Leu 625 630635 640 Glu Pro Glu Val Glu Asn Arg Ala Pro Glu Gly Asp Thr Val Ser His645 650 655 Glu Gly Ser Glu Arg Ala Thr Pro Thr Lys Ser Glu Asp Thr GlyAsp 660 665 670 Val Ser Ser Arg Asp Glu Thr Pro Arg Tyr Arg Arg Ser SerSer Val 675 680 685 Asn Met Lys Lys Met Gln Lys Met Phe Gln Asn Ala AlaGlu Glu Asn 690 695 700 Val Arg Asn Ile Arg Ala Tyr Val Thr Glu Leu LysGlu Arg Val Ala 705 710 715 720 Lys Leu Gln Tyr Gln Lys Gln Leu Leu ValCys Gln Val Leu Glu Leu 725 730 735 Glu Ser Asn Glu Gly Lys Thr Asn AspMet Glu Glu Asp Ser Glu Glu 740 745 750 Asn Ala Gly Ser Leu Gln Asp GlyPro Asp Ser Trp Asp Arg Leu Phe 755 760 765 Lys Glu Gln Met Gln His IleIle Gln Leu Trp Asp Leu Cys His Val 770 775 780 Ser Ile Ile His Arg ThrGln Phe Tyr Leu Leu Phe Arg Gly Asp Arg 785 790 795 800 Ala Asp Gln IleTyr Ile Glu Val Glu Val Arg Arg Leu Thr Trp Leu 805 810 815 Gln Gln HisPhe Ala Glu Val Gly Asp Ala Ser Pro Ala Ala Gly Asp 820 825 830 Asp SerThr Ile Ser Leu Ala Ser Ser Ile Lys Ala Leu Arg Asn Glu 835 840 845 ArgGlu Phe Leu Ala Arg Arg Met Gly Ser Arg Leu Thr Glu Glu Glu 850 855 860Arg Glu Arg Leu Phe Ile Lys Trp Gln Val Pro Leu Glu Ala Lys Gln 865 870875 880 Arg Lys Leu Gln Leu Val Asn Arg Leu Trp Thr Asp Pro Asn Asp Gln885 890 895 Ala His Ile Asp Glu Ser Ala Asp Ile Val Ala Arg Leu Val GlyPhe 900 905 910 Cys Glu Gly Gly Asn Ile Ser Lys Glu Met Phe Glu Leu AsnPhe Ala 915 920 925 Val Pro Ala Ser Arg Lys Pro Trp Leu Met Gly Trp GlnPro Ile Ser 930 935 940 Asn Met Ile Arg Glu Lys Thr Gln Leu Trp 945 9503 1004 PRT Arabidopsis thaliana 3 Met Thr Ile Lys Thr Pro Gly Thr ProVal Ser Lys Met Asp Arg Thr 1 5 10 15 Pro Ala Val Thr Pro Gly Gly SerSer Arg Ser Arg Glu Glu Lys Ile 20 25 30 Val Val Thr Val Arg Leu Arg ProMet Asn Lys Arg Glu Leu Leu Ala 35 40 45 Lys Asp Gln Val Ala Trp Glu CysVal Asn Asp His Thr Ile Val Ser 50 55 60 Lys Pro Gln Val Gln Glu Arg LeuHis His Gln Ser Ser Phe Thr Phe 65 70 75 80 Asp Lys Val Phe Gly Pro GluSer Leu Thr Glu Asn Val Tyr Glu Asp 85 90 95 Gly Val Lys Asn Val Ala LeuSer Ala Leu Met Gly Ile Asn Ala Thr 100 105 110 Ile Phe Ala Tyr Gly GlnThr Ser Ser Gly Lys Thr Tyr Thr Met Arg 115 120 125 Gly Val Thr Glu LysAla Val Asn Asp Ile Tyr Asn His Ile Ile Lys 130 135 140 Val Ser Thr AlaLeu Val Arg Val Asn Val Leu Asp Val Lys Cys Ser 145 150 155 160 Ser SerAsp Gln Tyr Leu Tyr Leu Phe His Cys Cys Phe Gln Thr Pro 165 170 175 GluArg Asp Phe Thr Ile Lys Ile Ser Gly Leu Glu Ile Tyr Asn Glu 180 185 190Asn Val Arg Asp Leu Leu Asn Ser Asp Ser Gly Arg Ala Leu Lys Leu 195 200205 Leu Asp Asp Pro Glu Lys Gly Thr Val Val Glu Lys Leu Val Glu Glu 210215 220 Thr Ala Asn Asn Asp Asn His Leu Arg His Leu Ile Ser Ile Cys Glu225 230 235 240 Ala Gln Arg Gln Val Gly Glu Thr Ala Leu Asn Asp Thr SerSer Arg 245 250 255 Ser His Gln Ile Ile Arg Leu Asn Phe Val Asp Leu AlaGly Ser Glu 260 265 270 Arg Ala Ser Gln Ser Gln Ala Asp Gly Thr Arg LeuArg Glu Gly Cys 275 280 285 His Ile Asn Leu Ser Leu Met Thr Leu Thr ThrVal Ile Arg Lys Leu 290 295 300 Arg Tyr Cys Thr Tyr Ile Phe Ser Glu ArgLeu Lys Ser Gln Ser Gln 305 310 315 320 Ile Leu Phe Asn Val Gly Lys ArgSer Gly His Ile Pro Tyr Arg Asp 325 330 335 Ser Lys Leu Thr Arg Ile LeuGln His Ser Leu Gly Gly Asn Ala Arg 340 345 350 Thr Ala Ile Ile Cys ThrLeu Ser Pro Ala Leu Ala His Val Glu Gln 355 360 365 Ser Arg Asn Thr LeuTyr Phe Ala Asn Arg Ala Lys Glu Val Thr Asn 370 375 380 Asn Ala His ValAsn Met Val Val Ser Asp Lys Gln Leu Val Lys His 385 390 395 400 Leu GlnLys Glu Val Ala Arg Leu Glu Ala Glu Arg Arg Thr Pro Gly 405 410 415 ProSer Thr Glu Lys Asp Phe Lys Ile Gln Gln Met Glu Met Glu Ile 420 425 430Gly Glu Leu Arg Arg Gln Arg Asp Asp Ala Gln Ile Gln Leu Glu Glu 435 440445 Leu Arg Gln Lys Leu Gln Gln Asp Gln Gln Gln Asn Lys Gly Leu Asn 450455 460 Pro Phe Glu Ser Pro Asp Pro Pro Val Arg Lys Cys Leu Ser Tyr Ser465 470 475 480 Val Ala Val Thr Pro Ser Ser Glu Asn Lys Arg Leu Asn ArgAsn Glu 485 490 495 Arg Ala Arg Lys Thr Thr Met Arg Gln Ser Met Ile ArgGln Ser Ser 500 505 510 Thr Ala Pro Phe Thr Leu Met His Glu Ile Arg LysLeu Glu His Leu 515 520 525 Gln Glu Gln Leu Gly Glu Glu Ala Thr Lys AlaLeu Glu Val Leu Gln 530 535 540 Lys Glu Val Ala Cys His Arg Leu Gly AsnGln Asp Ala Ala Gln Thr 545 550 555 560 Ile Ala Lys Leu Gln Ala Glu IleArg Glu Met Arg Thr Val Lys Pro 565 570 575 Ser Ala Met Leu Lys Glu ValGly Asp Val Ile Ala Pro Asn Lys Ser 580 585 590 Val Ser Ala Asn Leu LysGlu Glu Ile Thr Arg Leu His Ser Gln Gly 595 600 605 Ser Thr Ile Ala AsnLeu Glu Glu Gln Leu Glu Ser Val Gln Lys Ser 610 615 620 Ile Asp Lys LeuVal Met Ser Leu Pro Ser Asn Ile Ser Ala Gly Asp 625 630 635 640 Glu ThrPro Lys Thr Lys Asn His His His Gln Ser Lys Lys Lys Lys 645 650 655 LeuLeu Pro Leu Thr Pro Ala Ser Ala Ser Asn Arg Gln Asn Phe Leu 660 665 670Lys Ser Pro Cys Ser Pro Leu Ser Ala Ser Arg Gln Val Leu Asp Cys 675 680685 Asp Ala Glu Asn Lys Ala Pro Gln Glu Asn Asn Ser Ser Ala Ala Arg 690695 700 Gly Ala Thr Thr Pro Gln Gly Ser Glu Lys Glu Thr Pro Gln Lys Gly705 710 715 720 Glu Glu Ser Gly Asp Val Ser Ser Arg Glu Gly Thr Pro GlyTyr Arg 725 730 735 Arg Ser Ser Ser Val Asn Met Lys Lys Met Gln Gln MetPhe Gln Asn 740 745 750 Ala Ala Glu Glu Asn Val Arg Ser Ile Arg Ala TyrVal Thr Glu Leu 755 760 765 Lys Glu Arg Val Ala Lys Leu Gln Tyr Gln LysGln Leu Leu Val Cys 770 775 780 Gln Val Leu Glu Leu Glu Ala Asn Asp GlyAla Gly Tyr Ser Val Glu 785 790 795 800 Asn Glu Glu Asn Thr Ile Met GluAsp Glu Glu Gln Asn Gln Val Ala 805 810 815 Trp His Ile Thr Phe Ile GluGlu Arg Gln Gln Ile Ile Glu Leu Trp 820 825 830 His Val Cys His Val SerIle Ile His Arg Thr Gln Phe Tyr Leu Leu 835 840 845 Phe Lys Gly Asp GlnAla Asp Gly Gln Ile Tyr Met Glu Val Glu Leu 850 855 860 Arg Arg Leu ThrTrp Leu Glu Gln His Leu Ala Glu Val Gly Asn Ala 865 870 875 880 Thr ProAla Arg Asn Cys Asp Glu Ser Val Val Ser Leu Ser Ser Ser 885 890 895 IleLys Ala Leu Arg Arg Glu Arg Glu Phe Leu Ala Lys Arg Val Asn 900 905 910Ser Arg Leu Thr Pro Glu Glu Arg Glu Glu Leu Tyr Met Lys Trp Asp 915 920925 Val Pro Leu Glu Gly Lys Gln Arg Lys Leu Gln Phe Val Asn Lys Leu 930935 940 Trp Thr Asp Pro Tyr Asp Ser Arg His Val Gln Glu Ser Ala Glu Ile945 950 955 960 Val Ala Lys Leu Val Gly Phe Cys Glu Ser Gly Asn Ile SerLys Glu 965 970 975 Met Phe Glu Leu Asn Phe Ala Val Pro Ser Asp Lys ArgGln Trp Asn 980 985 990 Ile Gly Trp Asp Asn Ile Ser Asn Leu Leu His Leu995 1000 4 4590 DNA Arabidopsis thaliana 4 atgggacctc cgagaactccgttaagtaag atagataagt ccaatcctta tactccatgt 60 ggttccaaag ttacagaggagaagattctt gtcactgttc ggatgagacc attaaattgg 120 agggaacatg ccaaatatgatctcattgct tgggaatgtc ccgatgatga gactatcgtc 180 tttaagaatc cgaatcctgacaaggctcca acaaaatatt catttggtat tttttattag 240 aatctagtcg attgcttattttgtcttctc ttctttccat ttttgttata tagctcattt 300 aagttttttc ttctttgtacttatagataa agtttttgag ccaacttgtg caactcaaga 360 ggtgtatgaa ggtggttctagggacgttgc actatctgca ttagcaggaa caaatggtaa 420 ctacacagaa atctaaaaaatcccattatc taaggatatg ttatttgtgg atatgtgatt 480 tcttgtctct atctattgcagcaactattt ttgcttatgg tcagactagt agtggcaaga 540 cgtttacaat gaggggggttactgaaagtg ttgttaaaga tatatacgaa catataagaa 600 aagtgagtga tcaaatggatgtgttttggt tttggttttg ttacgaagtg atatgttctt 660 tttctattct catggatctgttgaaatcta tgtagactca agaaaggagc tttgtcttga 720 aagtatctgc tttggagatttataacgaga ccgtggttga ccttttaaat cgtgatactg 780 ggccccttag actgttagatgatccagagg taagtaaagg gtcggaaaca ctaagtcaag 840 gctcatgaac tatctcggccagtataagaa tgctgtgtat attgcagaaa ggaaccattg 900 tggaaaatct ggtggaggaagtggtagaaa gcaggcaaca tttacagcat ttaattagca 960 tttgtgaagg taattcttctatcacctgtt aagatgctct atatcttttt atgcatttga 1020 tggtgatagt attctaagtaatgtttagca aagccttaga accatgtaga tgacatatat 1080 aaaatatgac taattatttcactaatcact aatgagacat atgatatcat cattttattt 1140 cctgttatgt gattttccatttattgttac agatcaaagg caagtaggtg aaactgcttt 1200 aaacgacaaa agttcaagatcgcatcagat tattcggctg gtatgatcta tacttatctg 1260 atatgctgca tgattgaattaatattctat taaacatgga gaagatagtt ttgcctagtt 1320 atttgctacc attagacagtaaaagtaacc acttcccata atttctcatg ttctctgctt 1380 ttaatattca gaccatccatagcagtctca gggaaatagc aggctgtgtg cagtctttca 1440 tggcaactct ggtacgtcatttattatcat tgggttgaag cttcacttgg tattgacgac 1500 attttaattg gtgcagaatcttgttgatct tgctggaagc gaacgtgctt ttcaaacaaa 1560 cgcagatggt ctaagattgaaggaaggaag ccacattaac cgtagtttat tgacacttac 1620 tacagtgatc agaaaactgaggtcagaaag cacactagtt tgaacaccat gattgtcttt 1680 ggcatgtcta aattaccttttagattgatg aacattttca cctgtagggc atcttaagaa 1740 attagcaaaa cattaaagtttttaattatt tctgttattt tggttgaagt tctggaagaa 1800 aaagggatca cgtgccgtatagagactcaa agctgacaag aattttgcaa aattcacttg 1860 gcgggaatgc tagaacagcaattatatgta cgattagtcc ggcactgagt catgttgaac 1920 agacaaaaaa gacactttcttttgccatga gtgcgaagga agtcaccaat tgcgctaaag 1980 taaacatggt atgtcacttagttaaatatt tgtagaggtc tttcgttttg cagaaccagg 2040 atgattgtat agagctttgtctttggctta ctaaagcttt tctttggcct ttcattgcaa 2100 atgtggtttt gtttctgagtagcaatcatc atcctatagt gagcatatca tatgcaatta 2160 tctgcctgca tgtctattctgctcttgcca tgaattaaga atacaaaatg catcgcgtta 2220 cctgtaatga tgtagtgagatatattttca aagccttctt ttgtcgctct cccactaaca 2280 ttactccttt tgcttcaatcttctttgtgt ttgatgaatc acaaaacggt tttgcttgtt 2340 cttgatagaa agtttacccctctgctggta ttcaatttag ggatgtgaat ttagttgatt 2400 ttctgtcgta ggttgtctcagaaaaaaagc tgctgaaaca tctgcagcaa aaagtagcta 2460 aactcgagtc agagttaagaagcccggagc ccagttcatc cacctgctta aagtctctgt 2520 taattgagaa agagatgaaaatccaacagg tatgacttag actttaagaa ctagaattca 2580 catcttgaat tatctcttctgaatatttta aatgtggagt ccacagacta ggtaaaccat 2640 atgggacgtg aacttaagtttattaacaaa aacaacttga attttttatc acttgcaatt 2700 attcattcat gtttaactaatggcagatgg aatccgaaat gaaagagcta aagcgtcaga 2760 gagacattgc acagtctgaacttgatctag aaagaaaagc aaaggagcgg aaggtatgtg 2820 cgctgctaat atgatataccaaaattttct gggataactt caaccttatg cagacatgcc 2880 tatataattg gttgcagggatcaagtgagt gtgaaccttt ttctcaagtg gcaaggtgtc 2940 tctcatacca tacaaaggaggaatccattc caagcaaatc tgttccgtca agccgcagaa 3000 cagctcgtga taggagaaaggataatgtga ggcagtcttt aacttcggcg gatccaacag 3060 cgctagtcca agaaattcgtttgcttgaaa aacatcaaaa gaagttgggt gaagaagcaa 3120 accaagcact ggatctaattcacaaagaag tgacatccca taaactgggt gatcaacaag 3180 ctgcagaaaa agttgcgaaaatgctgtcag aaatcaggga catgcagaag agcaaccttt 3240 taactgagga aattgtagttggtgataaag ccaacctgaa ggaagaaata aatcggttga 3300 attctcaaga aattgcagctttggagaaga agctagagtg tgttcagaac actattgaca 3360 tgcttgtctc gtcttttcagacagatgaac agactccaga tttcaggacc caggtgaaaa 3420 agaagagact tcttcccttcggcttaagta atagtcctaa cctacagcat atgatccggg 3480 gaccatgttc acctctttctgggactgaaa ataaggatcc tgagagcaat gttgtgtctg 3540 ctaattcagc cccagtgtcttttggagcta ctccaccaaa gagggatgat aaccgatgtc 3600 gtacacagtc aagggaaggaacgcctgtat cacgacaagc aaattcagtc gatattaaga 3660 gaatgaatcg aatgtataagaatgctgccg aagagaatat acgtaatatc aaatcttatg 3720 ttactggatt gaaagaacgtgtggcgaagc ttcaatacca gaagcagctg cttgtttgcc 3780 aggtcagttt acacttgtgacttatgctca ttcatttaca aatttgttca tcagtaatca 3840 gtaacttaaa aagcttgtacatgttaggtg ttagaactgg aggcaaacga gacaggcgct 3900 gcttcggagt acgatgcaaccgatgaatct cagatggatt ggccattgtg ttttgaagag 3960 cagagaaagc aaatcataatgttatggcat ctgtgccaca tttcaatcat tcacagaaca 4020 caattctaca tgttattcaaaggagaccca gctgatcaaa tctacatgga ggttgagctc 4080 cgcaggctga catggctcgaacagcacttg gctgagcttg ggaatgcgag tcctgcattg 4140 cttggtgatg aaccagcaagctacgtagca tcgaggtagt acaagagctt tccttttctt 4200 caattctatg agttaaaaccatttttcact agcatatata tgtctttggt gtgctgcagt 4260 ataagagctt tgaagcaggagagagagtat ttggcgaaac gggtaaacac aaagctagga 4320 gcagaggaaa gagagatgctatacctgaaa tgggatgtcc cgccggtggg aaaacagagg 4380 agacaacagt tcatcaacaaactctggaca gatcctcaca acatgcaaca cgtgagagag 4440 agtgcggaga tcgtggctaagcttgtcgga ttctgtgact cgggagagac cataaggaaa 4500 gaaatgtttg agctcaactttgcatctcct tcagataaga agacttggat gatgggttgg 4560 aacttcatat ccaacttgttgcatctctag 4590 5 4583 DNA Artificial sequence TES-1 MUTANT SEQUENCE 5atgggacctc cgagaactcc gttaagtaag atagataagt ccaatcctta tactccatgt 60ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagacc attaaattgg 120agggaacatg ccaaatatga tctcattgct tgggaatgtc ccgatgatga gactatcgtc 180tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtat tttttattag 240aatctagtcg attgcttatt ttgtcttctc ttctttccat ttttgttata tagctcattt 300aagttttttc ttctttgtac ttatagataa agtttttgag ccaacttgtg caactcaaga 360ggtgtatgaa ggtggttcta gggacgttgc actatctgca ttagcaggaa caaatggtaa 420ctacacagaa atctaaaaaa tcccattatc taaggatatg ttatttgtgg atatgtgatt 480tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagt agtggcaaga 540cgtttacaat gaggggggtt actgaaagtg ttgttaaaga tatatacgaa catataagaa 600aagtgagtga tcaaatggat gtgttttggt tttggttttg ttacgaagtg atatgttctt 660tttctattct catggatctg ttgaaatcta tgtagactca agaaaggagc tttgtcttga 720aagtatctgc tttggagatt tataacgaga ccgtggttga ccttttaaat cgtgatactg 780ggccccttag actgttagat gatccagagg taagtaaagg gtcggaaaca ctaagtcaag 840gctcatgaac tatctcggcc agtataagaa tgctgtgtat attgcagaaa ggaaccattg 900tggaaaatct ggtggaggaa gtggtagaaa gcaggcaaca tttacagcat ttaattagca 960tttgtgaagg taattcttct atcacctgtt aagatgctct atatcttttt atgcatttga 1020tggtgatagt attctaagta atgtttagca aagccttaga accatgtaga tgacatatat 1080aaaatatgac taattatttc actaatcact aatgagacat atgatatcat cattttattt 1140cctgttatgt gattttccat ttattgttac agatcaaagg caagtaggtg aaactgcttt 1200aaacgacaaa agttcaagat cgcatcagat tattcggctg gtatgatcta tacttatctg 1260atatgctgca tgattgaatt aatattctat taaacatgga gaagatagtt ttgcctagtt 1320atttgctacc attagacagt aaaagtaacc acttcccata atttctcatg ttctctgctt 1380ttaatattca gaccatccat agcagtctca gggaaatagc aggctgtgtg cagtctttca 1440tggcaactct ggtacgtcat ttattatcat tgggttgaag cttcacttgg tattgacgac 1500attttaattg gtgcagaatc ttgttgatct tgctggaagc gaacgtgctt ttcaaacaaa 1560cgcagatggt ctaagattga aggaaggaag ccacattaac cgtagtttat tgacacttac 1620tacagtgatc agaaaactga ggtcagaaag cacactagtt tgaacaccat gattgtcttt 1680ggcatgtcta aattaccttt tagattgatg aacattttca cctgtagggc atcttaagaa 1740attagcaaaa cattaaagtt tttaattatt tctgttattt tggttgaagt tctggaagaa 1800aaagggatca cgtgccgtat agagactcaa agctgacaag aattttgcaa aattcacttg 1860gcgggaatgc tagaacagca attatatgta cgattagtcc ggcactgagt catgttgaac 1920agacaaaaaa gacactttct tttgccatga gtgcgaagga agtcaccaat tgcgctaaag 1980taaacatggt atgtcactta gttaaatatt tgtagaggtc tttcgttttg cagaaccagg 2040atgattgtat agagctttgt ctttggctta ctaaagcttt tctttggcct ttcattgcaa 2100atgtggtttt gtttctgagt agcaatcatc atcctatagt gagcatatca tatgcaatta 2160tctgcctgca tgtctattct gctcttgcca tgaattaaga atacaaaatg catcgcgtta 2220cctgtaatga tgtagtgaga tatattttca aagccttctt ttgtcgctct cccactaaca 2280ttactccttt tgcttcaatc ttctttgtgt ttgatgaatc acaaaacggt tttgcttgtt 2340cttgatagaa agtttacccc tctgctggta ttcaatttag ggatgtgaat ttagttgatt 2400ttctgtcgta ggttgtctca gaaaaaaagc tgctgaaaca tctgcagcaa aaagtagcta 2460aactcgagtc agagttaaga agcccggagc ccagttcatc cacctgctta aagtctctgt 2520taattgagaa agagatgaaa atccaacagg tatgacttag actttaagaa ctagaattca 2580catcttgaat tatctcttct gaatatttta aatgtggagt ccacagacta ggtaaaccat 2640atgggacgtg aacttaagtt tattaacaaa aacaacttga attttttatc acttgcaatt 2700attcattcat gtttaactaa tggcagatgg aatccgaaat gaaagagcta aagcgtcaga 2760gagacattgc acagtctgaa cttgatctag aaagaaaagc aaaggagcgg aaggtatgtg 2820cgctgctaat atgatatacc aaaattttct gggataactt caaccttatg cagacatgcc 2880tatataattg gttgcaggga tcaagtgagt gtgaaccttt ttctcaagtg gcaaggtgtc 2940tctcatacca tacaaaggag gaatccattc caagcaaatc tgttccgtca agccgcagaa 3000cagctcgtga taggagaaag gataatgtga ggcagtcttt aacttcggcg gatccaacag 3060cgctagtcca agaaattcgt ttgcttgaaa aacatcaaaa gaagttgggt gaagaagcaa 3120accaagcact ggatctaatt cacaaagaag tgacatccca taaactgggt gatcaacaag 3180ctgcagaaaa agttgcgaaa atgctgtcag aaatcaggga catgcagaag agcaaccttt 3240taactgagga aattgtagtt ggtgataaag ccaacctgaa ggaagaaata aatcggttga 3300attctcaaga aattgcagct ttggagaaga agctagagtg tgttcagaac actattgaca 3360tgcttgtctc gtcttttcag acagatgaac agactccaga tttcaggacc caggtgaaaa 3420agaagagact tcttcccttc ggcttaagta atagtcctaa cctacagcat atgatccggg 3480gaccatgttc acctctttct gggactgaaa ataaggatcc tgagagcaat gttgtgtctg 3540ctaattcagc cccagtgtct tttggagcta ctccaccaaa gagggatgat aaccgatgtc 3600gtacacagtc aagggaagga acgcctgtat cacgacaagc aaattcagtc gatattaaga 3660gaatgaatcg aatgtataag aatgctgccg aagagaatat acgtaatatc aaatcttatg 3720ttactggatt gaaagaacgt gtggcgaagc ttcaatacca gaagcagctg cttgtttgcc 3780aggtcagttt acacttgtga cttatgctca ttcatttaca aatttgttca tcagtaatca 3840gtaacttaaa aagcttgtac atgttaggtg ttagaactgg aggcaaacga gacaggcgct 3900gcttcggagt acgatgcaac cgatgaatct cagatggatt ggccattgtg ttttgaagag 3960cagagaaagc aaatcataat gttatggcat ctgtgccaca tttcaatcat tcacagaaca 4020caattctaca tgttattcaa aggagaccca gctgatcaaa tctacatgga ggttgagctc 4080cgcaggctga catggctcga acagcacttg gctgagcttg ggaatgcgag tcctgcattg 4140cttggtgatg aaccagcaag ctacgtagca tcgaggtagt acaagagctt tccttttctt 4200caattctatg agttaaaacc atttttcact agcatatata tgtctttggt gtgctgcagt 4260ataagagctt tgaagcagga gagagagtat ttggcgaaac gggtaaacac aaagctagga 4320gcagaggaaa gagagatgct atacctgaaa tgggatgtcc cgccggtggg aaaacagagg 4380agacaacagt tcatcaacaa actctggaca gatcctcaca acatgcaaca cgtgagagag 4440agtgcggaga tcgtggctaa gcttgtcgga ttctgtgact cgggagagac cataaggaaa 4500gaaatgtcaa ctttgcatct ccttcagata agaagacttg gatgatgggt tggaacttca 4560tatccaactt gttgcatctc tag 4583 6 4570 DNA Artificial sequence WS2.R TESSEQUENCE 6 atgggacctc cgagaactcc gttaagtaag atagataagt ccaatccttatactccatgt 60 ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagaccattaaattgg 120 agggaacatg ccaaatatga tctcattgct tgggaatgtc ctgatgatgaaactatcgtc 180 tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtatttttattaga 240 atctagtcaa ttgcttattt gtcttctctt ctttccaatt ttgttatatagctcatttaa 300 gtttttgtct tgtttgtact tacagataaa gtttttgagc caacttgtgccactcaagag 360 gtgtatgaag gtggttctag ggacgttgca ctatctgcat tagcaggaacaaatggtaaa 420 tacacagata tctaaaaaaa tcccattatc taaggatatt ttatttgtggatatgtgatt 480 tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagtagtggcaaga 540 cgtttacaat gaggggggtt acggaaagtg ttgttaaaga tatatacgaacatataagaa 600 aagtgagtga ccaaatggat gtgttttggt tttggttttg ttacgaagtgatatgttctt 660 tttctatact catggatctg ttgaaatcta tgtagactca agaaaggagctttgtcttga 720 aagtatctgc tttggagatc tataatgaga ccgtggttga ccttttaaatcgtgatactg 780 ggccccttcg actgttagat gatccagagg taagtaaagg gtcggaaacactaggctcat 840 gaactatctc ggccagtata agaatgttgt gtatgttgca gaaaggaaccattgtggaaa 900 atctggtgga ggaagtggta gaaagcaggc aacatttaca gcatttaattagcatttgtg 960 aaggtaattc ttctatcacc tgttaagatg ctctatatct ctttatgcatttgatgatga 1020 tagtattcta agtcatgttt agcaaagcct tagaaccatg tagatgacatatataaaata 1080 tgactcatta tttcactaat cactaatgag acatatgata tcatctttttatttcctgtt 1140 atgtgatttt ccatttattg tacagatcaa aggcaagtag gtgaaactgctttaaacgac 1200 aaaagttcaa gatcgcatca gattattcgg ctggtatgat ctatactcatctgatatgct 1260 gcataattga attaatattc tattaaacat ggagaagata gttttgcctagttatttgct 1320 accattagac agtaaaagta accactttcc ataatttctc atgttctctgcttttaatat 1380 tcagaccatc catagcagtc tcagggaaat agcaggctgt gtgcagtctttcatggcaac 1440 tctggtacgt catttattat catagggttg aagcttcact tggtattgacgacattttaa 1500 ttggtgcaga atcttgttga tcttgctgga agcgaacgtg cttttcaaacaaacgcagat 1560 ggtctaagac tgaaggaagg aagccacatt aaccgtagtt tattgacacttactacagtg 1620 atcagaaaac tgaggtcaga aagcacacta gtctgaacac catgattatctttggcatgt 1680 ctaaattacc ttttaggttg atgaacattt tcacccgtag ggcaccttaagaaattagca 1740 aaacatttaa ttatttctgt tattttggtg aagttctgga agaaaaagggatcacgtgcc 1800 atatagagac tcaaagctga caagaatttt gcaaaattca cttggcgggaatgctagaac 1860 agcaattatc tgtacgatta gcccagcact gagtcacgtt gaacagacaaaaaagacact 1920 ctcttttgcc atgagtgcga aggaagtcac caactgcgct aaagtaaacatggtatgtca 1980 cttagttaaa tatttggaga ggtctttcgt tttgcagaaa caggatgattgtttggagct 2040 ttgtctttgg cttcctaaag cttttctttg gtctttcatt gaaaatgtggtttgtttgtg 2100 agtagcaatc atcatcctat agtgagcata tcatatgcaa ttatctgcctgcatagctat 2160 tctgctcttc ccatgaatta aggatacaaa atgcatcgcg ttacctgtaatgatgtagta 2220 agatataatt caaagccttc tttgtcgctc tcccactaac attactccttttgcttcaat 2280 cttctttgtg tttgatgaat aacaaaacgg ttttgcttgt tcttgatagaaagtttaccc 2340 ctctactggt attcaattta gggatgtgaa tttagttgat tttctgttgtaggttgtctc 2400 agaaaaaaag ctgctgaaac atctgcagca aaaagtagct aaactcgagtcagagttaag 2460 aagcccggag cccagttcat ccacctgctt aaagtctctg ttaattgagaaagagatgaa 2520 aatccaacag gtatgactta gactttaaga actagaattc acatcttgaattatctcttc 2580 tgaatatttt aaatgtggag tccacagact aggtaaacca tatgggacgtgaacttaagt 2640 ttattaacaa aaacaacttg aattttttat gacttgcaat tattcattcatgtttaacta 2700 atggcagatg gaatccgaaa tgaaagagct aaagcgtcag agagacattgcacagtctga 2760 acttgatcta gaaagaaaag caaaggagcg gaaggtatgt gcgctgctaatatgatatac 2820 caaaattttc tgggataact tcaaccttat gcagacatgc ctatataattggttgcaggg 2880 atcaagtgag tgtgaacctt tttctcaagt ggcaaggtgt ctctcataccatacaaagga 2940 ggaatccatt ccaagcaaat ctgttccgtc aagccgcaga acagctcgtgataggagaaa 3000 ggataatgtg aggcagtctt taacttcagc ggatccaaca gcgctagtccaagaaattcg 3060 tttgcttgaa aaacatcaaa agaagttggg tgaagaagca aaccaagcactggatctaat 3120 tcacaaagaa gtgacatccc ataaactggg tgatcaacaa gctgcagagaaagttgcgaa 3180 aatgctatca gaaatcaggg acatgcagaa gagcaacctt ttaactgaggaaattgtagt 3240 tggtgataaa gctaacctga aggaagaaat aaatcgtttg aattctcaagaaattgcagc 3300 tttggagaag aagctagagt gtgttcagaa cactattgac atgcttgtctcgtcttttca 3360 gacagatgaa cagactccag atttcaggac ccaggtgaaa aagaagagacttcttccctt 3420 cggcttaagt aatagtccta acctacagca tatgatccgg ggaccatgttcacctctttc 3480 tgggactgaa aataaggatc ctgagagcaa tgttgtgtct gctaattcaatcccagtgtc 3540 ttttggagct actccaccaa agagggatga taaccgatgt cgtacaccgtcaagggaagg 3600 aacgcctgta tcacgacaag caaattcagt cgatattaag agaatgaatcgaatgtataa 3660 gaatgctgcc gaagaaaata tacgtaacat caaatcttat gttactggattgaaagaacg 3720 tgtggcgaag cttcaatacc agaagcagct tcttgtttgc caggtcagtttacacttgtg 3780 acttatgctc attcatttac aaatttgttc atcaggaatc agtaacttaaaagcttgcac 3840 atgtataggt gttagaactg gaggcaaacg agacaggcgc tgcttcggagtacgatgcaa 3900 ccgatgaatc tcggatggac tggccattgt gttttgaaga gcagagaaagcaaatcataa 3960 tgttatggca tctgtgccac atttcaatca ttcacagaac acaattctacatgttattca 4020 aaggagaccc agctgatcaa atctacatgg aagttgagct ccgcaggctgacatggctcg 4080 aacagcactt ggctgagctt gggaatgcga gtcctgcatt gcttggtgatgaaccagcaa 4140 gctacgtagc atcaaggtag tacaagagct ttccttttct tcaattctatgagttaaacc 4200 atttttcaca agcatatata tgtctttggt gtgctgcagt ataagagctttgaagcagga 4260 aagagagtat ttggcgaaac gggtaaacac aaagctagga gcagaggaaagagagatgct 4320 atacctgaaa tgggatgtcc cgccggtggg aaaacagagg aggcaacagttcatcaacaa 4380 actctggaca gatcctcaca acatgcaaca cgtgagagag agtgcggagatcgtggctaa 4440 gcttgtcgga ttctgtgact cgggagagac cataaggaaa gaaatgtttgagctcaactt 4500 tgcatctcct tcagataaga agacatggat gatgggttgg aacttcatatccaacttgtt 4560 gcatctctag 4570 7 4560 DNA Artificial sequence TES-4MUTANT SEQUENCE 7 atgggacctc cgagaactcc gttaagtaag atagataagt ccaatccttatactccatgt 60 ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagaccattaaattgg 120 agggaacatg ccaaatatga tctcattgct tgggaatgtc ctgatgatgaaactatcgtc 180 tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtatttttattaga 240 atctagtcaa ttgcttattt gtcttctctt ctttccaatt ttgttatatagctcatttaa 300 gtttttgtct tgtttgtact tacagataaa gtttttgagc caacttgtgccactcaagag 360 gtgtatgaag gtggttctag ggacgttgca ctatctgcat tagcaggaacaaatggtaaa 420 tacacagata tctaaaaaaa tcccattatc taaggatatt ttatttgtggatatgtgatt 480 tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagtagtggcaaga 540 cgtttacaat gaggggggtt acggaaagtg ttgttaaaga tatatacgaacatataagaa 600 aagtgagtga ccaaatggat gtgttttggt tttggttttg ttacgaagtgatatgttctt 660 tttctatact catggatctg ttgaaatcta tgtagactca agaaaggagctttgtcttga 720 aagtatctgc tttggagatc tataatgaga ccgtggttga ccttttaaatcgtgatactg 780 ggccccttcg actgttagat gatccagagg taagtaaagg gtcggaaacactaggctcat 840 gaactatctc ggccagtata agaatgttgt gtatgttgca gaaaggaaccattgtggaaa 900 atctggtgga ggaagtggta gaaagcaggc aacatttaca gcatttaattagcatttgtg 960 aaggtaattc ttctatcacc tgttaagatg ctctatatct ctttatgcatttgatgatga 1020 tagtattcta agtcatgttt agcaaagcct tagaaccatg tagatgacatatataaaata 1080 tgactcatta tttcactaat cactaatgag acatatgata tcatctttttatttcctgtt 1140 atgtgatttt ccatttattg tacagatcaa aggcaagtag gtgaaactgctttaaacgac 1200 aaaagttcaa gatcgcatca gattattcgg ctggtatgat ctatactcatctgatatgct 1260 gcataattga attaatattc tattaaacat ggagaagata gttttgcctagttatttgct 1320 accattagac agtaaaagta accactttcc ataatttctc atgttctctgcttttaatat 1380 tcagaccatc catagcagtc tcagggaaat agcaggctgt gtgcagtctttcatggcaac 1440 tctggtacgt catttattat catagggttg aagcttcact tggtattgacgacattttaa 1500 ttggtgcaga atcttgttga tcttgctgga agcgaacgtg cttttcaaacaaacgcagat 1560 ggtctaagac tgaaggaagg aagccacatt aaccgtagtt tattgacacttactacagtg 1620 atcagaaaac tgaggtcaga aagcacacta gtctgaacac catgattatctttggcatgt 1680 ctaaattacc ttttaggttg atgaacattt tcacccgtag ggcaccttaagaaattagca 1740 aaacatttaa ttatttctgt tattttggtg aagttctgga agaaaaagggatcacgtgcc 1800 atatagagac tcaaagctga caagaatttt gcaaaattca cttggcgggaatgctagaac 1860 agcaattatc tgtacgatta gcccagcact gagtcacgtt gaacagacaaaaaagacact 1920 ctcttttgcc atgagtgcga aggaagtcac caactgcgct aaagtaaacatggtatgtca 1980 cttagttaaa tatttggaga ggtctttcgt tttgcagaaa caggatgattgtttggagct 2040 ttgtctttgg cttcctaaag cttttctttg gtctttcatt gaaaatgtggtttgtttgtg 2100 agtagcaatc atcatcctat agtgagcata tcatatgcaa ttatctgcctgcatagctat 2160 tctgctcttc ccatgaatta aggatacaaa atgcatcgcg ttacctgtaatgatgtagta 2220 agatataatt caaagccttc tttgtcgctc tcccactaac attactccttttgcttcaat 2280 cttctttgtg tttgatgaat aacaaaacgg ttttgcttgt tcttgatagaaagtttaccc 2340 ctctactggt attcaattta gggatgtgaa tttagttgat tttctgttgtaggttgtctc 2400 agaaaaaaag ctgctgaaac atctgcagca aaaagtagct aaactcgagtcagagttaag 2460 aagcccggag cccagttcat ccacctgctt aaagtctctg ttaattgagaaagagatgaa 2520 aatccaacag gtatgactta gactttaaga actagaattc acatcttgaattatctcttc 2580 tgaatatttt aaatgtggag tccacagact aggtaaacca tatgggacgtgaacttaagt 2640 ttattaacaa aaacaacttg aattttttat gacttgcaat tattcattcatgtttaacta 2700 atggcagatg gaatccgaaa tgaaagagct aaagcgtcag agagacattgcacagtctga 2760 acttgatcta gaaagaaaag caaaggagcg gaaggtatgt gcgctgctaatatgatatac 2820 caaaattttc tgggataact tcaaccttat gcagacatgc ctatataattggttgcaggg 2880 atcaagtgag tgtgaacctt tttctcaagt ggcaaggtgt ctctcataccatacaaagga 2940 ggaatccatt ccaagcaaat ctgttccgtc aagccgcaga acagctcgtgataggagaaa 3000 ggataatgtg aggcagtctt taacttcagc ggatccaaca gcgctagtccaagaaattcg 3060 tttgcttgaa aaacatcaaa agaagttggg tgaagaagca aaccaagcactggatctaat 3120 tcacaaagaa gtgacatccc ataaactggg tgatcaacaa gctgcagagaaagttgcgaa 3180 aatgctatca gaaatcaggg acatgcagaa gagcaacctt ttaactgaggaaattgtagt 3240 tggtgataaa gctaacctga aggaagaaat aaatcgtttg aattctcaagaaattgcagc 3300 tttggagaag aagctagagt gtgttcagaa cactattgac attcttttcagacagatgaa 3360 cagactccag atttcaggac ccaggtgaaa aagaagagac ttcttcccttcggcttaagt 3420 aatagtccta acctacagca tatgatccgg ggaccatgtt cacctctttctgggactgaa 3480 aataaggatc ctgagagcaa tgttgtgtct gctaattcaa tcccagtgtcttttggagct 3540 actccaccaa agagggatga taaccgatgt cgtacaccgt caagggaaggaacgcctgta 3600 tcacgacaag caaattcagt cgatattaag agaatgaatc gaatgtataagaatgctgcc 3660 gaagaaaata tacgtaacat caaatcttat gttactggat tgaaagaacgtgtggcgaag 3720 cttcaatacc agaagcagct tcttgtttgc caggtcagtt tacacttgtgacttatgctc 3780 attcatttac aaatttgttc atcaggaatc agtaacttaa aagcttgcacatgtataggt 3840 gttagaactg gaggcaaacg agacaggcgc tgcttcggag tacgatgcaaccgatgaatc 3900 tcggatggac tggccattgt gttttgaaga gcagagaaag caaatcataatgttatggca 3960 tctgtgccac atttcaatca ttcacagaac acaattctac atgttattcaaaggagaccc 4020 agctgatcaa atctacatgg aagttgagct ccgcaggctg acatggctcgaacagcactt 4080 ggctgagctt gggaatgcga gtcctgcatt gcttggtgat gaaccagcaagctacgtagc 4140 atcaaggtag tacaagagct ttccttttct tcaattctat gagttaaaccatttttcaca 4200 agcatatata tgtctttggt gtgctgcagt ataagagctt tgaagcaggaaagagagtat 4260 ttggcgaaac gggtaaacac aaagctagga gcagaggaaa gagagatgctatacctgaaa 4320 tgggatgtcc cgccggtggg aaaacagagg aggcaacagt tcatcaacaaactctggaca 4380 gatcctcaca acatgcaaca cgtgagagag agtgcggaga tcgtggctaagcttgtcgga 4440 ttctgtgact cgggagagac cataaggaaa gaaatgtttg agctcaactttgcatctcct 4500 tcagataaga agacatggat gatgggttgg aacttcatat ccaacttgttgcatctctag 4560 8 4570 DNA Artificial sequence LER-R TES SEQUENCE 8atgggacctc cgagaactcc gttaagtaag atagataagt ccaatcctta tactccatgt 60ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagacc attaaattgg 120agggaacatg ccaaatatga tctcattgct tgggaatgtc ctgatgatga aactatcgtc 180tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtat ttttattaga 240atctagtcaa ttgcttattt gtcttctctt ctttccaatt ttgttatata gctcatttaa 300gtttttgtct tgtttgtact tacagataaa gtttttgagc caacttgtgc cactcaagag 360gtgtatgaag gtggttctag ggacgttgca ctatctgcat tagcaggaac aaatggtaaa 420tacacagata tctaaaaaaa tcccattatc taaggatatt ttatttgtgg atatgtgatt 480tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagt agtggcaaga 540cgtttacaat gaggggggtt acggaaagtg ttgttaaaga tatatacgaa catataagaa 600aagtgagtga ccaaatggat gtgttttggt tttggttttg ttacgaagtg atatgttctt 660tttctatact catggatctg ttgaaatcta tgtagactca agaaaggagc tttgtcttga 720aagtatctgc tttggagatc tataatgaga ccgtggttga ccttttaaat cgtgatactg 780ggccccttcg actgttagat gatccagagg taagtaaagg gtcggaaaca ctaggctcat 840gaactatctc ggccagtata agaatgttgt gtatgttgca gaaaggaacc attgtggaaa 900atctggtgga ggaagtggta gaaagcaggc aacatttaca gcatttaatt agcatttgtg 960aaggtaattc ttctatcacc tgttaagatg ctctatatct ctttatgcat ttgatgatga 1020tagtattcta agtcatgttt agcaaagcct tagaaccatg tagatgacat atataaaata 1080tgactcatta tttcactaat cactaatgag acatatgata tcatcttttt atttcctgtt 1140atgtgatttt ccatttattg tacagatcaa aggcaagtag gtgaaactgc tttaaacgac 1200aaaagttcaa gatcgcatca gattattcgg ctggtatgat ctatactcat ctgatatgct 1260gcataattga attaatattc tattaaacat ggagaagata gttttgccta gttatttgct 1320accattagac agtaaaagta accactttcc ataatttctc atgttctctg cttttaatat 1380tcagaccatc catagcagtc tcagggaaat agcaggctgt gtgcagtctt tcatggcaac 1440tctggtacgt catttattat catagggttg aagcttcact tggtattgac gacattttaa 1500ttggtgcaga atcttgttga tcttgctgga agcgaacgtg cttttcaaac aaacgcagat 1560ggtctaagac tgaaggaagg aagccacatt aaccgtagtt tattgacact tactacagtg 1620atcagaaaac tgaggtcaga aagcacacta gtctgaacac catgattatc tttggcatgt 1680ctaaattacc ttttaggttg atgaacattt tcacccgtag ggcaccttaa gaaattagca 1740aaacatttaa ttatttctgt tattttggtg aagttctgga agaaaaaggg atcacgtgcc 1800atatagagac tcaaagctga caagaatttt gcaaaattca cttggcggga atgctagaac 1860agcaattatc tgtacgatta gcccagcact gagtcacgtt gaacagacaa aaaagacact 1920ctcttttgcc atgagtgcga aggaagtcac caactgcgct aaagtaaaca tggtatgtca 1980cttagttaaa tatttggaga ggtctttcgt tttgcagaaa caggatgatt gtttggagct 2040ttgtctttgg cttcctaaag cttttctttg gtctttcatt gaaaatgtgg tttgtttgtg 2100agtagcaatc atcatcctat agtgagcata tcatatgcaa ttatctgcct gcatagctat 2160tctgctcttc ccatgaatta aggatacaaa atgcatcgcg ttacctgtaa tgatgtagta 2220agatataatt caaagccttc tttgtcgctc tcccactaac attactcctt ttgcttcaat 2280cttctttgtg tttgatgaat aacaaaacgg ttttgcttgt tcttgataga aagtttaccc 2340ctctactggt attcaattta gggatgtgaa tttagttgat tttctgttgt aggttgtctc 2400agaaaaaaag ctgctgaaac atctgcagca aaaagtagct aaactcgagt cagagttaag 2460aagcccggag cccagttcat ccacctgctt aaagtctctg ttaattgaga aagagatgaa 2520aatccaacag gtatgactta gactttaaga actagaattc acatcttgaa ttatctcttc 2580tgaatatttt aaatgtggag tccacagact aggtaaacca tatgggacgt gaacttaagt 2640ttattaacaa aaacaacttg aattttttat gacttgcaat tattcattca tgtttaacta 2700atggcagatg gaatccgaaa tgaaagagct aaagcgtcag agagacattg cacagtctga 2760acttgatcta gaaagaaaag caaaggagcg gaaggtatgt gcgctgctaa tatgatatac 2820caaaattttc tgggataact tcaaccttat gcagacatgc ctatataatt ggttgcaggg 2880atcaagtgag tgtgaacctt tttctcaagt ggcaaggtgt ctctcatacc atacaaagga 2940ggaatccatt ccaagcaaat ctgttccgtc aagccgcaga acagctcgtg ataggagaaa 3000ggataatgtg aggcagtctt taacttcagc ggatccaaca gcgctagtcc aagaaattcg 3060tttgcttgaa aaacatcaaa agaagttggg tgaagaagca aaccaagcac tggatctaat 3120tcacaaagaa gtgacatccc ataaactggg tgatcaacaa gctgcagaga aagttgcgaa 3180aatgctatca gaaatcaggg acatgcagaa gagcaacctt ttaactgagg aaattgtagt 3240tggtgataaa gctaacctga aggaagaaat aaatcgtttg aattctcaag aaattgcagc 3300tttggagaag aagctagagt gtgttcagaa cactattgac atgcttgtct cgtcttttca 3360gacagatgaa cagactccag atttcaggac ccaggtgaaa aagaagagac ttcttccctt 3420cggcttaagt aatagtccta acctacagca tatgatccgg ggaccatgtt cacctctttc 3480tgggactgaa aataaggatc ctgagagcaa tgttgtgtct gctaattcaa tcccagtgtc 3540ttttggagct actccaccaa agagggatga taaccgatgt cgtacaccgt caagggaagg 3600aacgcctgta tcacgacaag caaattcagt cgatattaag agaatgaatc gaatgtataa 3660gaatgctgcc gaagaaaata tacgtaacat caaatcttat gttactggat tgaaagaacg 3720tgtggcgaag cttcaatacc agaagcagct tcttgtttgc caggtcagtt tacacttgtg 3780acttatgctc attcatttac aaatttgttc atcaggaatc agtaacttaa aagcttgcac 3840atgtataggt gttagaactg gaggcaaacg agacaggcgc tgcttcggag tacgatgcaa 3900ccgatgaatc tcggatggac tggccattgt gttttgaaga gcagagaaag caaatcataa 3960tgttatggca tctgtgccac atttcaatca ttcacagaac acaattctac atgttattca 4020aaggagaccc agctgatcaa atctacatgg aagttgagct ccgcaggctg acatggctcg 4080aacagcactt ggctgagctt gggaatgcga gtcctgcatt gcttggtgat gaaccagcaa 4140gctacgtagc atcaaggtag tacaagagct ttccttttct tcaattctat gagttaaacc 4200atttttcaca agcatatata tgtctttggt gtgctgcagt ataagagctt tgaagcagga 4260aagagagtat ttggcgaaac gggtaaacac aaagctagga gcagaggaaa gagagatgct 4320atacctgaaa tgggatgtcc cgccggtggg aaaacagagg aggcaacagt tcatcaacaa 4380actctggaca gatcctcaca acatgcaaca cgtgagagag agtgcggaga tcgtggctaa 4440gcttgtcgga ttctgtgact cgggagagac cataaggaaa gaaatgtttg agctcaactt 4500tgcatctcct tcagataaga agacatggat gatgggttgg aacttcatat ccaacttgtt 4560gcatctctag 4570 9 4570 DNA Artificial sequence TES-3 MUTANT SEQUENCE 9atgggacctc cgagaactcc gttaagtaag atagataagt ccaatcctta tactccatgt 60ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagacc attaaattgg 120agggaacatg ccaaatatga tctcattgct tgggaatgtc ctgatgatga aactatcgtc 180tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtat ttttattaga 240atctagtcaa ttgcttattt gtcttctctt ctttccaatt ttgttatata gctcatttaa 300gtttttgtct tgtttgtact tacagataaa gtttttgagc caacttgtgc cactcaagag 360gtgtatgaag gtggttctag ggacgttgca ctatctgcat tagcaggaac aaatggtaaa 420tacacagata tctaaaaaaa tcccattatc taaggatatt ttatttgtgg atatgtgatt 480tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagt agtggcaaga 540cgtttacaat gaggggggtt acggaaagtg ttgttaaaga tatatacgaa catataagaa 600aagtgagtga ccaaatggat gtgttttggt tttggttttg ttacgaagtg atatgttctt 660tttctatact catggatctg ttgaaatcta tgtagactca agaaaggagc tttgtcttga 720aagtatctgc tttggagatc tataatgaga ccgtggttga ccttttaaat cgtgatactg 780ggccccttcg actgttagat gatccagagg taagtaaagg gtcggaaaca ctaggctcat 840gaactatctc ggccagtata agaatgttgt gtatgttgca gaaaggaacc attgtggaaa 900atctggtgga ggaagtggta gaaagcaggc aacatttaca gcatttaatt agcatttgtg 960aaggtaattc ttctatcacc tgttaagatg ctctatatct ctttatgcat ttgatgatga 1020tagtattcta agtcatgttt agcaaagcct tagaaccatg tagatgacat atataaaata 1080tgactcatta tttcactaat cactaatgag acatatgata tcatcttttt atttcctgtt 1140atgtgatttt ccatttattg tacagatcaa aggcaagtag gtgaaactgc tttaaacgac 1200aaaagttcaa gatcgcatca gattattcgg ctggtatgat ctatactcat ctgatatgct 1260gcataattga attaatattc tattaaacat ggagaagata gttttgccta gttatttgct 1320accattagac agtaaaagta accactttcc ataatttctc atgttctctg cttttaatat 1380tcagaccatc catagcagtc tcagggaaat agcaggctgt gtgcagtctt tcatggcaac 1440tctggtacgt catttattat catagggttg aagcttcact tggtattgac gacattttaa 1500ttggtgcaga atcttgttga tcttgctgga agcgaacgtg cttttcaaac aaacgcagat 1560ggtctaagac tgaaggaagg aagccacatt aaccgtagtt tattgacact tactacagtg 1620atcagaaaac tgaggtcaga aagcacacta gtctgaacac catgattatc tttggcatgt 1680ctaaattacc ttttaggttg atgaacattt tcacccgtag ggcaccttaa gaaattagca 1740aaacatttaa ttatttctgt tattttggtg aagttctgga agaaaaaggg atcacgtgcc 1800atatagagac tcaaagctga caagaatttt gcaaaattca cttggcggga atgctagaac 1860agcaattatc tgtacgatta gcccagcact gagtcacgtt gaacagacaa aaaagacact 1920ctcttttgcc atgagtgcga aggaagtcac caactgcgct aaagtaaaca tggtatgtca 1980cttagttaaa tatttggaga ggtctttcgt tttgcagaaa caggatgatt gtttggagct 2040ttgtctttgg cttcctaaag cttttctttg gtctttcatt gaaaatgtgg tttgtttgtg 2100agtagcaatc atcatcctat agtgagcata tcatatgcaa ttatctgcct gcatagctat 2160tctgctcttc ccatgaatta aggatacaaa atgcatcgcg ttacctgtaa tgatgtagta 2220agatataatt caaagccttc tttgtcgctc tcccactaac attactcctt ttgcttcaat 2280cttctttgtg tttgatgaat aacaaaacgg ttttgcttgt tcttgataga aagtttaccc 2340ctctactggt attcaattta gggatgtgaa tttagttgat tttctgttgt aggttgtctc 2400agaaaaaaag ctgctgaaac atctgcagca aaaagtagct aaactcgagt cagagttaag 2460aagcccggag cccagttcat ccacctgctt aaagtctctg ttaattgaga aagagatgaa 2520aatccaacag gtatgactta gactttaaga actagaattc acatcttgaa ttatctcttc 2580tgaatatttt aaatgtggag tccacagact aggtaaacca tatgggacgt gaacttaagt 2640ttattaacaa aaacaacttg aattttttat gacttgcaat tattcattca tgtttaacta 2700atggcagatg gaatccgaaa tgaaagagct aaagcgtcag agagacattg cacagtctga 2760acttgatcta gaaagaaaag caaaggagcg gaaggtatgt gcgctgctaa tatgatatac 2820caaaattttc tgggataact tcaaccttat gcagacatgc ctatataatt ggttgcaggg 2880atcaagtgag tgtgaacctt tttctcaagt ggcaaggtgt ctctcatacc atacaaagga 2940ggaatccatt ccaagcaaat ctgttccgtc aagccgcaga acagctcgtg ataggagaaa 3000ggataatgtg aggcagtctt taacttcagc ggatccaaca gcgctagtcc aagaaattcg 3060tttgcttgaa aaacatcaaa agaagttggg tgaagaagca aaccaagcac tggatctaat 3120tcacaaagaa gtgacatccc ataaactggg tgatcaacaa gctgcagaga aagttgcgaa 3180aatgctatca gaaatcaggg acatgcagaa gagcaacctt ttaactgagg aaattgtagt 3240tggtgataaa gctaacctga aggaagaaat aaatcgtttg aattctcaag aaattgcagc 3300tttggagaag aagctagagt gtgttcagaa cactattgac atgcttgtct cgtcttttca 3360gacagatgaa cagactccag atttcaggac ccaggtgaaa aagaagagac ttcttccctt 3420cggcttaagt aatagtccta acctacagca tatgatccgg ggaccatgtt cacctctttc 3480tgggactgaa aataaggatc ctgagagcaa tgttgtgtct gctaattcaa tcccagtgtc 3540ttttggagct actccaccaa agagggatga taactgatgt cgtacaccgt caagggaagg 3600aacgcctgta tcacgacaag caaattcagt cgatattaag agaatgaatc gaatgtataa 3660gaatgctgcc gaagaaaata tacgtaacat caaatcttat gttactggat tgaaagaacg 3720tgtggcgaag cttcaatacc agaagcagct tcttgtttgc caggtcagtt tacacttgtg 3780acttatgctc attcatttac aaatttgttc atcaggaatc agtaacttaa aagcttgcac 3840atgtataggt gttagaactg gaggcaaacg agacaggcgc tgcttcggag tacgatgcaa 3900ccgatgaatc tcggatggac tggccattgt gttttgaaga gcagagaaag caaatcataa 3960tgttatggca tctgtgccac atttcaatca ttcacagaac acaattctac atgttattca 4020aaggagaccc agctgatcaa atctacatgg aagttgagct ccgcaggctg acatggctcg 4080aacagcactt ggctgagctt gggaatgcga gtcctgcatt gcttggtgat gaaccagcaa 4140gctacgtagc atcaaggtag tacaagagct ttccttttct tcaattctat gagttaaacc 4200atttttcaca agcatatata tgtctttggt gtgctgcagt ataagagctt tgaagcagga 4260aagagagtat ttggcgaaac gggtaaacac aaagctagga gcagaggaaa gagagatgct 4320atacctgaaa tgggatgtcc cgccggtggg aaaacagagg aggcaacagt tcatcaacaa 4380actctggaca gatcctcaca acatgcaaca cgtgagagag agtgcggaga tcgtggctaa 4440gcttgtcgga ttctgtgact cgggagagac cataaggaaa gaaatgtttg agctcaactt 4500tgcatctcct tcagataaga agacatggat gatgggttgg aacttcatat ccaacttgtt 4560gcatctctag 4570 10 4570 DNA Artificial sequence STUD- MUTANT SEQUENCE 10atgggacctc cgagaactcc gttaagtaag atagataagt ccaatcctta tactccatgt 60ggttccaaag ttacagagga gaagattctt gtcactgttc ggatgagacc attaaattgg 120agggaacatg ccaaatatga tctcattgct tgggaatgtc ctgatgatga aactatcgtc 180tttaagaatc cgaatcctga caaggctcca acaaaatatt catttggtat ttttattaga 240atctagtcaa ttgcttattt gtcttctctt ctttccaatt ttgttatata gctcatttaa 300gtttttgtct tgtttgtact tacagataaa gtttttgagc caacttgtgc cactcaagag 360gtgtatgaag gtggttctag ggacgttgca ctatctgcat tagcaggaac aaatggtaaa 420tacacagata tctaaaaaaa tcccattatc taaggatatt ttatttgtgg atatgtgatt 480tcttgtctct atctattgca gcaactattt ttgcttatgg tcagactagt agtggcaaga 540cgtttacaat gaggggggtt acggaaagtg ttgttaaaga tatatacgaa catataagaa 600aagtgagtga ccaaatggat gtgttttggt tttggttttg ttacgaagtg atatgttctt 660tttctatact catggatctg ttgaaatcta tgtagactca agaaaggagc tttgtcttga 720aagtatctgc tttggagatc tataatgaga ccgtggttga ccttttaaat cgtgatactg 780ggccccttcg actgttagat gatccagagg taagtaaagg gtcggaaaca ctaggctcat 840gaactatctc ggccagtata agaatgttgt gtatgttgca gaaaggaacc attgtggaaa 900atctggtgga ggaagtggta gaaagcaggc aacatttaca gcatttaatt agcatttgtg 960aaggtaattc ttctatcacc tgttaagatg ctctatatct ctttatgcat ttgatgatga 1020tagtattcta agtcatgttt agcaaagcct tagaaccatg tagatgacat atataaaata 1080tgactcatta tttcactaat cactaatgag acatatgata tcatcttttt atttcctgtt 1140atgtgatttt ccatttattg tacagatcaa aggcaagtag gtgaaactgc tttaaacgac 1200aaaagttcaa gatcgcatca gattattcgg ctggtatgat ctatactcat ctgatatgct 1260gcataattga attaatattc tattaaacat ggagaagata gttttgccta gttatttgct 1320accattagac agtaaaagta accactttcc ataatttctc atgttctctg cttttaatat 1380tcagaccatc catagcagtc tcagggaaat agcaggctgt gtgcagtctt tcatggcaac 1440tctggtacgt catttattat catagggttg aagcttcact tggtattgac gacattttaa 1500ttggtgcaga atcttgttga tcttgctgga agcgaacgtg cttttcaaac aaacgcagat 1560ggtctaagac tgaaggaagg aagccacatt aaccgtagtt tattgacact tactacagtg 1620atcagaaaac tgaggtcaga aagcacacta gtctgaacac catgattatc tttggcatgt 1680ctaaattacc ttttaggttg atgaacattt tcacccgtag ggcaccttaa gaaattagca 1740aaacatttaa ttatttctgt tattttggtg aagttctgga agaaaaaggg atcacgtgcc 1800atatagagac tcaaagctga caagaatttt gcaaaattca cttggcggga atgctagaac 1860agcaattatc tgtacgatta gcccagcact gagtcacgtt gaacagacaa aaaagacact 1920ctcttttgcc atgagtgcga aggaagtcac caactgcgct aaagtaaaca tggtatgtca 1980cttagttaaa tatttggaga ggtctttcgt tttgcagaaa caggatgatt gtttggagct 2040ttgtctttgg cttcctaaag cttttctttg gtctttcatt gaaaatgtgg tttgtttgtg 2100agtagcaatc atcatcctat agtgagcata tcatatgcaa ttatctgcct gcatagctat 2160tctgctcttc ccatgaatta aggatacaaa atgcatcgcg ttacctgtaa tgatgtagta 2220agatataatt caaagccttc tttgtcgctc tcccactaac attactcctt ttgcttcaat 2280cttctttgtg tttgatgaat aacaaaacgg ttttgcttgt tcttgataga aagtttaccc 2340ctctactggt attcaattta gggatgtgaa tttagttgat tttctgttgt aggttgtctc 2400agaaaaaaag ctgctgaaac atctgcagca aaaagtagct aaactcgagt cagagttaag 2460aagcccggag cccagttcat ccacctgctt aaagtctctg ttaattgaga aagagatgaa 2520aatccaacag gtatgactta gactttaaga actagaattc acatcttgaa ttatctcttc 2580tgaatatttt aaatgtggag tccacagact aggtaaacca tatgggacgt gaacttaagt 2640ttattaacaa aaacaacttg aattttttat gacttgcaat tattcattca tgtttaacta 2700atggcagatg gaatccgaaa tgaaagagct aaagcgtcag agagacattg cacagtctga 2760acttgatcta gaaagaaaag caaaggagcg gaaggtatgt gcgctgctaa tatgatatac 2820caaaattttc tgggataact tcaaccttat gcagacatgc ctatataatt ggttgcaggg 2880atcaagtgag tgtgaacctt tttctcaagt ggcaaggtgt ctctcatacc atacaaagga 2940ggaatccatt ccaagcaaat ctgttccgtc aagccgcaga acagctcgtg ataggagaaa 3000ggataatgtg aggcagtctt taacttcagc ggatccaaca gcgctagtcc aagaaattcg 3060tttgcttgaa aaacatcaaa agaagttggg tgaagaagca aaccaagcac tggatctaat 3120tcacaaagaa gtgacatccc ataaactggg tgatcaacaa gctgcagaga aagttgcgaa 3180aatgctatca gaaatcaggg acatgcagaa gagcaacctt ttaactgagg aaattgtagt 3240tggtgataaa gctaacctga aggaagaaat aaatcgtttg aattcttaag aaattgcagc 3300tttggagaag aagctagagt gtgttcagaa cactattgac atgcttgtct cgtcttttca 3360gacagatgaa cagactccag atttcaggac ccaggtgaaa aagaagagac ttcttccctt 3420cggcttaagt aatagtccta acctacagca tatgatccgg ggaccatgtt cacctctttc 3480tgggactgaa aataaggatc ctgagagcaa tgttgtgtct gctaattcaa tcccagtgtc 3540ttttggagct actccaccaa agagggatga taaccgatgt cgtacaccgt caagggaagg 3600aacgcctgta tcacgacaag caaattcagt cgatattaag agaatgaatc gaatgtataa 3660gaatgctgcc gaagaaaata tacgtaacat caaatcttat gttactggat tgaaagaacg 3720tgtggcgaag cttcaatacc agaagcagct tcttgtttgc caggtcagtt tacacttgtg 3780acttatgctc attcatttac aaatttgttc atcaggaatc agtaacttaa aagcttgcac 3840atgtataggt gttagaactg gaggcaaacg agacaggcgc tgcttcggag tacgatgcaa 3900ccgatgaatc tcggatggac tggccattgt gttttgaaga gcagagaaag caaatcataa 3960tgttatggca tctgtgccac atttcaatca ttcacagaac acaattctac atgttattca 4020aaggagaccc agctgatcaa atctacatgg aagttgagct ccgcaggctg acatggctcg 4080aacagcactt ggctgagctt gggaatgcga gtcctgcatt gcttggtgat gaaccagcaa 4140gctacgtagc atcaaggtag tacaagagct ttccttttct tcaattctat gagttaaacc 4200atttttcaca agcatatata tgtctttggt gtgctgcagt ataagagctt tgaagcagga 4260aagagagtat ttggcgaaac gggtaaacac aaagctagga gcagaggaaa gagagatgct 4320atacctgaaa tgggatgtcc cgccggtggg aaaacagagg aggcaacagt tcatcaacaa 4380actctggaca gatcctcaca acatgcaaca cgtgagagag agtgcggaga tcgtggctaa 4440gcttgtcgga ttctgtgact cgggagagac cataaggaaa gaaatgtttg agctcaactt 4500tgcatctcct tcagataaga agacatggat gatgggttgg aacttcatat ccaacttgtt 4560gcatctctag 4570

1. A plant in which, by virtue of modulation of the expression of aTETRASPORE (TES) or TES-like (TLK) gene, or of modulation of theactivity of a protein encoded by such a gene, tetrad formation in theanther is disrupted, or is capable of being disrupted, such that callosecross-wall formation in the tetrad fails to the extent that fusionbetween two or more of the four microspore nuclei may occur.
 2. A plantaccording to claim 1 wherein fusion between two or more of the tetradnuclei results in the formation of sperm of greater than half the ploidyof its somatic cells.
 3. A plant according to claim 1 or 2 whereinfusion between the nuclei of the tetrad results in the formation ofsperm of heterogeneous ploidy.
 4. A plant according to claim 2 or 3wherein at least some of said sperm are diploid or triploid.
 5. A plantaccording to any one of the preceding claims wherein modulation of saidTES or TLK gene is by virtue of a transgenic construct which providesfor expression of antisense RNA or RNAi that suppresses the TES or TLKgene.
 6. A plant according to any one of the preceding claims whereinmodulation of said TES or TLK gene is inducible.
 7. A plant according toclaim 6 wherein modulation is inducible by virtue of a transgenicconstruct as defined in claim 5 wherein expression of the antisense RNAor RNAi is under the control of an inducible promoter.
 8. A plantaccording to any one of the preceding claims wherein said TES or TLKgene: (a) encodes a polypeptide sequence as set out in SEQ ID NO: 1, 2or 3; or (b) has a cDNA sequence having 60% or more homology to asequence of (a); or (c) has a cDNA sequence capable of hybridisingselectively to the complement of a sequence of (a); and which gene, whenfunctional, encodes a kinesin protein; and, when non-functional, causesthe plant to fail to form callose cross-walls in the tetrad.
 9. A plantaccording to any one of the preceding claims which is an ornamentalplant, or an arable crop plant.
 10. A progeny plant of a plant of anyone of the preceding claims.
 11. A method of obtaining a progeny plantfrom male and female parent plants whose somatic cells have differentgenome ploidy, said method comprising (a) providing as a male parent aplant according to any one of claims 1 to 9; (b) providing as a femaleparent a plant whose ploidy differs from the plant of (a); (c) allowingthe male parent of (a) to produce pollen containing sperm of greaterthan half the ploidy of its somatic cells, and optionally ofheterogeneous ploidy; (d) allowing the male parent of (a) to pollinatethe female parent of (b); and recovering a seed that results from saidpollination; and optionally (e) growing up a progeny plant from saidseed.
 12. A method of obtaining a progeny plant from male and femaleparent plants whose somatic cells have different genome ploidy, saidmethod comprising producing a plant according to any one of claims 1 to9; and (a) providing said plant as a male parent plant; (b) providing asthe female parent a plant whose ploidy differs from the plant of (a);(c) allowing the male parent of (a) to produce pollen containing spermof greater than half the ploidy of its somatic cells, and optionally ofheterogeneous ploidy; (d) allowing the male parent of (a) to pollinatethe female parent of (b); and recovering a seed that results from saidpollination; and optionally (e) growing up a progeny plant from saidseed.
 13. A method according to claim 12 wherein said male parent plantis produced by transformation with a construct according to claim 5, orby inactivating a TES or TLK gene by mutation.
 14. A method according toclaim 12 or 13 wherein the male and female parent plants are of: (i)different varieties, subspecies, lines or cultivars within the samespecies; or (ii) different species within the same genus; or (iii)different species in different genera.
 15. Use of a plant according toany one of claims 1 to 9 in a breeding programme.
 16. An TESpolynucleotide which: (a) encodes a polypeptide sequence as set out inSEQ ID NO: 1; or (b) has a cDNA sequence having 60% or more homology toa sequence of (a); or (c) has a cDNA sequence capable of hybridisingselectively to the complement of a sequence of (a); and encodes akinesin
 17. A polynucleotide sequence capable of hybridising selectivelyto all or part of the mRNA expressed from a gene as defined in claim 8and thus achieving antisense inhibition of said gene.
 18. A vectorcomprising a polynucleotide according to claim 16 or 17 operably linkedto a promoter.
 19. A vector according to claim 18 wherein the promoteris stage-specific and/or tissue specific and/or inducible.
 20. A vectoraccording to claim 18 or 19 wherein the polynucleotide is an antisensepolynucleotide according to claim 17 and the promoter is an induciblepromoter.
 21. A cell transformed with a polynucleotide sequenceaccording to claim 16 or 20 or a vector according to claim 18, 19 or 20.22. A cell according to claim 21 which is a plant cell.
 23. A method ofmaking a cell according to claim 21 or 22 comprising transforming a cellwith a polynucleotide sequence according to claim 16 or 17 or a vectoraccording to claim 18, 19 or
 20. 24. A method according to claim 23further comprising regenerating a plant from said transformed cell, andoptionally further propagating said plant to give progeny plants offirst, second or subsequent generations.
 25. A polypeptide encoded by apolynucleotide according to claim
 16. 26. A polypeptide according toclaim 25 having the sequence of SEQ ID NO:
 1. 27. An antibody whichspecifically recognises a polypeptide according to claim 25 or
 26. 28. Aprimer, or pair of primers, capable of amplifying a polynucleotide asdefined in claim 16 or 17 in a Polymerase Chain Reaction.
 29. A labelledprobe capable of selectively hybridising to a polynucleotide as definedin claim 16 or 17 and thus detecting said polynucleotide.